Step-function channelrhodopsins for optical control of cells

ABSTRACT

The invention, in some aspects relates to light-activated ion channel molecules and methods for their use to alter cell activity and function. Light-activated ion channel molecules of the invention can be administered to subjects, expressed in cells, and activated with light, to alter membrane potential in the cells, and can be used in methods for assaying compounds, treating diseases and conditions, compound screening and more.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional application Ser. No. 62/556,616 filed Sep. 11, 2017, thedisclosure of which is incorporated by reference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under grants2-R01-DA029639-5, 1-R24-MH106075 and 1-R01-NS087950 from the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention, in some aspects relates to compositions and methods foraltering conductance across membranes, cell activity, and cell function,also relates to the use of exogenous light-activated ion channels incells and subjects.

BACKGROUND OF THE INVENTION

Altering and controlling cell membrane and subcellular region ionpermeability has permitted examination of characteristics of cells,tissues, and organisms. Light-driven pumps and channels have been usedto silence or enhance cell activity. Molecular-genetic methods forpreparing cells that can be activated (e.g., depolarized) or inactivated(e.g., hyperpolarized) by specific wavelengths of light have beendeveloped (see, for example, Han, X. and E. S. Boyden, 2007, PLoS ONE 2,e299). Previously identified light-activated pumps and channels may berestricted to activation by particular wavelengths of light,localization, functional speed, etc. thus limiting their usefulness.

SUMMARY OF THE INVENTION

According to an aspect of the invention, light-activated ion channelpolypeptides are provided that include an amino acid sequence set forthas SEQ ID NO: 1 or a functional variant thereof. In some embodiments, afunctional variant of SEQ ID NO: 1 includes the amino acid sequence setforth as SEQ ID NO: 1 with 1, 2, 3, 4, or more amino acid sequencemodifications, wherein a Serine (S) is present at the amino acidposition that corresponds to amino acid 145 of SEQ ID NO: 1, and whereinthe light-activated ion-channel polypeptide has at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to amino acids 61-295 of SEQ ID NO: 1and at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to theremaining amino acids in the sequence set forth as SEQ ID NO: 1. Incertain embodiments, the amino acid sequence includes an Alanine (A) atthe position corresponding to amino acid 173 of SEQ ID NO: 1. In someembodiments, the light activated ion channel polypeptide is comprisesthe amino acid sequence set forth as SEQ ID NO: 3. In some embodiments,activating the ion channel comprises contacting the ion channelpolypeptide with one or more of a wavelength of a blue and a wavelengthof a green light. In certain embodiments, activating the light-activatedion channel polypeptide opens the channel of the light-activated ionchannel polypeptide. In some embodiments, contacting the light-activatedion channel polypeptide with a wavelength of blue or green light resultsin opening of the ion channel of the light-activated ion channelpolypeptide, wherein the channel remains in an open state for a timeperiod longer than an open state time period of a controllight-activated ion channel polypeptide. In some embodiments the lengthof the time period is statistically significantly longer time periodthan the control open state time period. In some embodiments, thecontrol light-activated ion channel polypeptide is a Chronos polypeptidecomprising the amino acid sequence set forth as SEQ ID NO: 6. In certainembodiments, the nucleic acid sequence encoding the light-activated ionchannel polypeptide comprises the nucleic acid sequence set forth as SEQID NO: 2. In some embodiments, the nucleic acid sequence encoding thelight-activated ion channel polypeptide comprises the nucleic acidsequence set forth as SEQ ID NO: 4. In certain embodiments, thelight-activated ion channel polypeptide is expressed in a membrane. Insome embodiments, the membrane is a cell membrane. In some embodiments,the light-activated ion channel polypeptide is expressed in a cell. Incertain embodiments, the cell is an excitable cell. In some embodiments,the cell is in a subject. In some embodiments, the membrane is a one ormore of a cell membrane of: a neuronal cell, a nervous system cell, acardiac cell, a circulatory system cell, a visual system cell, and anauditory system cell. In some embodiments, activating thelight-activated ion channel polypeptide alters the ion conductivity ofthe membrane in which the light-activated ion channel polypeptide isexpressed. In certain embodiments, activating the light-activated ionchannel polypeptide depolarizes the cell in which the light-activatedion channel polypeptide is expressed.

According to an aspect of the invention, fusion proteins that includethe light-activated ion channel polypeptide of any of the aforementionedembodiments or aspects of the invention are provided. In someembodiments, the fusion protein also includes one or more of atrafficking polypeptide, a signal polypeptide, an export polypeptide,and a detectable label polypeptide. In some embodiments, thelight-activated ion channel polypeptide comprises the amino acidsequence set forth as SEQ ID NO: 1, SEQ ID NO: 3, or a functionalvariant thereof.

According to another aspect of the invention, light-activated ionchannel polypeptides are provide that include an amino acid sequence setforth as SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 17 or SEQ ID NO: 18, or a functional variant thereof. In certainembodiments, the light-activated ion channel polypeptide comprises theamino acid sequence set forth as SEQ ID NO: 11 with 1, 2, 3, 4, or moreamino acid sequence modifications, wherein a Serine (S) is present atthe amino acid position that corresponds to amino acid 170 of SEQ ID NO:11, and wherein the light-activated ion-channel polypeptide has at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence setforth as SEQ ID NO: 11. In some embodiments, the amino acid sequenceincludes an alanine (A) at the amino acid position that corresponds toamino acid 198 of SEQ ID NO: 11. In some embodiments, thelight-activated ion channel comprises the amino acid sequence set forthas SEQ ID NO: 14 with 1, 2, 3, 4, or more amino acid sequencemodifications, wherein a Serine (S) is present at the amino acidposition that corresponds to amino acid 108 of SEQ ID NO: 14, andwherein the light-activated ion-channel polypeptide has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forthas SEQ ID NO: 14. In some embodiments, the amino acid sequence includesan alanine (A) at the amino acid position that corresponds to amino acid136 of SEQ ID NO: 14. In certain embodiments, the light-activated ionchannel polypeptide comprises the amino acid sequence set forth as SEQID NO: 17 with 1, 2, 3, 4, or more amino acid sequence modifications,wherein a Serine (S) is present at the amino acid position thatcorresponds to amino acid 165 of SEQ ID NO: 17, and wherein thelight-activated ion-channel polypeptide has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to the sequence set forth as SEQ ID NO: 17.In some embodiments, the amino acid sequence includes an alanine (A) atthe amino acid position that corresponds to amino acid 193 of SEQ ID NO:17. In certain embodiments, activating the light-activated ion channelpolypeptide opens the channel of the light-activated ion channelpolypeptide. In some embodiments, activating the ion channel polypeptidewith opens the ion channel of the light-activated ion channelpolypeptide, and wherein the channel remains in an open state for a timeperiod longer than an open state time period of a controllight-activated ion channel polypeptide. In some embodiments the lengthof the time period is a statistically significant longer time periodthan that of the control open state time period. In some embodiments,the control light-activated ion channel polypeptide is one of a Chrimsonpolypeptide comprising the amino acid sequence set forth as SEQ ID NO:10, a CoChR polypeptide comprising the amino acid sequence set forth asSEQ ID NO: 13, or a CsChR polypeptide comprising the amino acid sequenceset forth as SEQ ID NO: 16. In certain embodiments, the light-activatedion channel polypeptide is expressed in a membrane. In some embodiments,the membrane is a cell membrane. In some embodiments, thelight-activated ion channel polypeptide is expressed in a cell. Incertain embodiments, the cell is an excitable cell. In certainembodiments, the cell is in a subject. In some embodiments, the membraneis a one or more of a cell membrane of: a neuronal cell, a nervoussystem cell, a cardiac cell, a circulatory system cell, a visual systemcell, and an auditory system cell. In some embodiments, activating thelight-activated ion channel polypeptide alters the ion conductivity ofthe membrane in which the light-activated ion channel polypeptide isexpressed. In some embodiments, activating the light-activated ionchannel polypeptide depolarizes the cell in which the light-activatedion channel polypeptide is expressed.

According to another aspect of the invention, fusion proteins thatinclude any embodiment of an aforementioned aspect of the invention areprovided. In certain embodiments, the fusion protein also includes oneor more of a trafficking polypeptide, a signal polypeptide, an exportpolypeptide, and a detectable label polypeptide. In some embodiments,the light-activated ion channel polypeptide comprises the amino acidsequence set forth as SEQ ID NO: 11, 12, 14, 15, 17, 18, or a functionalvariant of thereof.

According to another aspect of the invention, polynucleotide moleculesare provide that include a nucleic acid sequence encoding alight-activated ion channel polypeptide of any embodiment of any of theaforementioned light-activated ion channel polypeptides. In someembodiments, the light-activated ion channel encoded by the nucleic acidsequence is expressed in a cell.

According to another aspect of the invention, vectors that include anyembodiment of any aforementioned nucleic acid sequence are provided. Incertain embodiments, the nucleic acid sequence is operatively linked toa promoter sequence. In some embodiments, the vector also includes one,two, or more nucleic acid signal sequences operatively linked to thenucleic acid sequence encoding the light-activated ion channelpolypeptide. In some embodiments, the vector is a plasmid vector, cosmidvector, viral vector, or an artificial chromosome. In some embodiments,the vector is in a cell. In certain embodiments, the cell is anexcitable cell. In some embodiments, the cell is a vertebrate cell. Insome embodiments, the cell is a mammalian cell. In certain embodiments,the cell is one or more of a neuronal cell, a nervous system cell, acardiac cell, a circulatory system cell, a visual system cell, and anauditory system cell.

According to another aspect of the invention, methods of altering ionconductivity of a membrane are provided, the methods including:expressing in a host membrane at least one of any of embodiment of anyaforementioned light-activated ion channel polypeptide and contactingthe at least one of the expressed light-activated ion channelpolypeptides with a light that activates at least one of thelight-activated ion channels and alters the ion conductivity of the hostmembrane. In some embodiments, the at least one expressedlight-activated ion channel polypeptides is a plurality of expressedlight-activated ion channel polypeptides. In some embodiments,activating the ion channel comprises contacting the ion channelpolypeptide with an activating wavelength of light. In some embodiments,the host membrane is in a cell. In certain embodiments, the cell is aneuronal cell and the method further comprises contacting thelight-activated ion channel polypeptide with a light under conditionssuitable to produce a spike in the neuronal cell. In some embodiments,activating the light-activated ion channel polypeptide opens the channelof the light-activated ion channel polypeptide. In some embodiments, thelight-activated ion channel polypeptide includes the amino acid sequenceset forth as SEQ ID NO: 1 with 1, 2, 3, 4, or more amino acid sequencemodifications, wherein a Serine (S) is present at the amino acidposition that corresponds to amino acid 145 of SEQ ID NO: 1, and whereinthe light-activated ion-channel polypeptide has at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to acids 61-295 of SEQ ID NO: 1 and atleast 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the remainingamino acids in the sequence set forth as SEQ ID NO: 1. In certainembodiments, the light-activated ion channel polypeptide has an Alanine(A) at the amino acid position corresponding to amino acid 173 of SEQ IDNO: 1. In some embodiments, the light activated ion channel polypeptidecomprises the amino acid sequence set forth as SEQ ID NO: 11 with 1, 2,3, 4, or more amino acid sequence modifications, wherein a Serine (S) ispresent at the amino acid position that corresponds to amino acid 170 ofSEQ ID NO: 11, and wherein the light-activated ion-channel polypeptidehas at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to thesequence set forth as SEQ ID NO: 11. In some embodiments, the amino acidsequence includes an alanine (A) at the amino acid position thatcorresponds to amino acid 198 of SEQ ID NO: 11. In certain embodiments,the light-activated ion channel polypeptide comprises the amino acidsequence set forth as SEQ ID NO: 14 with 1, 2, 3, 4, or more amino acidsequence modifications, wherein a Serine (S) is present at the aminoacid position that corresponds to amino acid 108 of SEQ ID NO: 14, andwherein the light-activated ion-channel polypeptide has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth asSEQ ID NO: 14. In some embodiments, the amino acid sequence includes analanine (A) at the amino acid position that corresponds to amino acid136 of SEQ ID NO: 14. In certain embodiments, the light-activated ionchannel polypeptide comprises the amino acid sequence set forth as SEQID NO: 17 with 1, 2, 3, 4, or more amino acid sequence modifications,wherein a Serine (S) is present at the amino acid position thatcorresponds to amino acid 165 of SEQ ID NO: 17, and wherein thelight-activated ion-channel polypeptide has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to the sequence set forth as SEQ ID NO: 17.In some embodiments, the amino acid sequence includes an alanine (A) atthe amino acid position that corresponds to amino acid 193 of SEQ ID NO:17. In some embodiments, the host membrane is a cell membrane. In someembodiments, the cell is an excitable cell. In certain embodiments, thecell is in a subject. In some embodiments, the host membrane is a cellmembrane of one or more of: a neuronal cell, a nervous system cell, acardiac cell, a circulatory system cell, a visual system cell, and anauditory system cell. In some embodiments, activating thelight-activated ion channel polypeptide alters the ion conductivity ofthe host membrane in which the light-activated ion channel polypeptideis expressed. In certain embodiments, activating the light-activated ionchannel polypeptide depolarizes the cell in which the light-activatedion channel polypeptide is expressed. In some embodiments, the cell is avertebrate cell. In some embodiments, the cell is a mammalian cell. Incertain embodiments, the cell is a human cell. In some embodiments, thecell comprises a plurality of the light-activated ion channelpolypeptides. In certain embodiments, the method also includescontacting the cell with a light that activates two or more of theplurality of the light-activated ion channel polypeptides. In someembodiments, contacting the light-activated ion channel polypeptide withan activating light results in opening of the ion channel of thelight-activated ion channel polypeptide, and wherein the channel remainsin an open state for a longer time period than an open state time periodof a control light-activated ion channel polypeptide. In someembodiments the length of the time period is a statistically significantlonger time period than that of the control open state time period. Incertain embodiments, the control light-activated ion channel polypeptideis a Chronos polypeptide comprising the amino acid sequence set forth asSEQ ID NO: 6, a Chrimson polypeptide comprising the amino acid sequenceset forth as SEQ ID NO: 10, a CoChR polypeptide comprising the aminoacid sequence set forth as SEQ ID NO: 13, or a CsChR polypeptidecomprising the amino acid sequence set forth as SEQ ID NO: 16. In someembodiments, expressing the light-activated ion channel polypeptide inthe host membrane includes administering to a cell that includes thehost membrane, a vector, wherein the vector includes a nucleic acidsequence encoding the light-activated ion channel and the administrationof the vector results in expression of the light-activated ion channelin the host membrane. In some embodiments, the vector further comprisesa signal sequence. In some embodiments, the vector also includes acell-specific promoter. In certain embodiments, depolarizing the cellmodulates a depolarization-mediated cell characteristic. In someembodiments, the depolarization-mediated cell characteristic is anaction potential. In some embodiments, the depolarization-mediated cellcharacteristic is release of a neurotransmitter. In certain embodiments,the amino acid sequence of the light-activated ion channel is set forthas SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18 or a functional variantthereof.

According to yet another aspect of the invention, methods of assessingthe effect of a candidate compound on ion conductivity of a membrane areprovided, the methods including: contacting a test membrane thatincludes the isolated light-activated ion channel polypeptide of anyembodiment of any of the aforementioned aspects with light underconditions suitable for altering ion conductivity of the membrane;contacting the test membrane with a candidate compound; and identifyingthe presence or absence of a change in ion conductivity of the membranecontacted with the light and the candidate compound compared to ionconductivity in a control cell contacted with the light and notcontacted with the candidate compound; wherein a change in the ionconductivity in the test membrane compared to the control indicates aneffect of the candidate compound on the ion conductivity of the testmembrane. In some embodiments, the membrane is in a cell. In someembodiments, altering the ion conductivity of the membrane depolarizesthe cell. In some embodiments, the change is an increase in ionconductivity of the membrane. In certain embodiments, the change is adecrease in ion conductivity of the membrane. In some embodiments, theeffect of the candidate compound is an effect on adepolarization-mediated cell characteristic in the test cell. In someembodiments, the method also includes characterizing the changeidentified in the depolarization or the depolarization-mediated cellcharacteristic. In certain embodiments, the depolarization-mediated cellcharacteristic is release of a neurotransmitter. In some embodiments,contacting the light-activated ion channel polypeptide with anactivating light results in opening of the ion channel of thelight-activated ion channel polypeptide, and wherein the channel remainsin an open state for a statistically significant longer time period thanan open state time period of a control light-activated ion channelpolypeptide. In certain embodiments, the control light-activated ionchannel polypeptide is a Chronos polypeptide comprising the amino acidsequence set forth as SEQ ID NO: 6, a Chrimson polypeptide comprisingthe amino acid sequence set forth as SEQ ID NO: 10, a CoChR polypeptidecomprising the amino acid sequence set forth as SEQ ID NO: 13, or aCsChR polypeptide comprising the amino acid sequence set forth as SEQ IDNO: 16. In some embodiments, expressing the light-activated ion channelpolypeptide in the test membrane comprises administering to a cell thatincludes the test membrane, a vector, wherein the vector comprises anucleic acid sequence encoding the light-activated ion channel and theadministration of the vector results in expression of thelight-activated ion channel in the test membrane. In some embodiments,the vector also includes a signal sequence. In certain embodiments, thevector also includes a cell-specific promoter. In some embodiments, theamino acid sequence of the light-activated ion channel is set forth asSEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, or a functional variantthereof.

According to another aspect of the invention, methods of treating adisorder in a subject are provided, the methods including administeringto a subject in need of such treatment, a therapeutically effectiveamount of a light-activated ion channel polypeptide of any embodiment ofany of the aforementioned aspects, to treat the disorder and contactingthe cell with light and activating the light-activated ion channel inthe cell under conditions sufficient to alter ion conductivity of a cellmembrane, wherein altering the conductivity of the cell membrane treatsthe disorder. In some embodiments, altering the ion conductivity of themembrane depolarizes the cell. In certain embodiments, the ionconductivity comprises one or more of ion flux and proton flux acrossthe light-activated ion channel polypeptide, or variant thereof. In someembodiments, the disease or condition is one or more of: a brain injury,a spinal cord injury, a nerve injury, epilepsy, a neurologicalcondition, an immune system disorder, a secretory system disorder, adegenerative neurological condition, cardiac dysfunction, vision loss,blindness, deafness, and hearing loss. In some embodiments, contactingthe light-activated ion channel polypeptide with an activating lightresults in opening of the ion channel of the light-activated ion channelpolypeptide, and wherein the channel remains in an open state for alonger time period than an open state time period of a controllight-activated ion channel polypeptide. In some embodiments the lengthof the time period is statistically significant longer time period thanthat of the control open state time period. In some embodiments, thecontrol light-activated ion channel polypeptide is a Chronos polypeptidethat includes the amino acid sequence set forth as SEQ ID NO: 6, aChrimson polypeptide that includes the amino acid sequence set forth asSEQ ID NO: 10, a CoChR polypeptide that includes the amino acid sequenceset forth as SEQ ID NO: 13, or a CsChR polypeptide that includes theamino acid sequence set forth as SEQ ID NO: 16. In certain embodiments,the light-activated ion channel is administered in the form of a cell,wherein the cell expresses the light-activated ion channel, or in theform of a vector, wherein the vector comprises a nucleic acid sequenceencoding the light-activated ion channel and the administration of thevector results in expression of the blue-light-activated ion channel ina cell in the subject. In some embodiments, the vector also includes asignal sequence. In some embodiments, the vector also includes acell-specific promoter. In certain embodiments, the method also includesadministering an additional therapeutic composition to the subject. Insome embodiments, depolarizing the cell modulates adepolarization-mediated cell characteristic. In some embodiments, thedepolarization-mediated cell characteristic is an action potential. Insome embodiments, the depolarization-mediated cell characteristic isrelease of a neurotransmitter. In certain embodiments, the amino acidsequence of the light-activated ion channel is set forth as SEQ ID NO:1, 3, 11, 12, 14, 15, 17, 18, or a functional variant thereof.

According to another aspect of the invention, light-activated ionchannel polypeptides are provided that include an amino acid sequenceset forth as SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or a functional variantthereof. In some embodiments, activating the light-activated ion channelpolypeptide opens the channel of the light-activated ion channelpolypeptide, and wherein activating the ion channel polypeptide withopens the ion channel of the light-activated ion channel polypeptide,and wherein the channel remains in an open state for a time periodsignificantly longer than an open state time period of a controllight-activated ion channel polypeptide. In certain embodiments, thecontrol light-activated ion channel polypeptide is one of a Chronospolypeptide comprising the amino acid sequence set forth as SEQ ID NO:6, a Chrimson polypeptide comprising the amino acid sequence set forthas SEQ ID NO: 10, a CoChR polypeptide comprising the amino acid sequenceset forth as SEQ ID NO: 13, or a CsChR polypeptide comprising the aminoacid sequence set forth as SEQ ID NO: 16. In some embodiments, thelight-activated ion channel polypeptide is expressed in a membrane, andoptionally the membrane is a cell membrane. In some embodiments, thelight-activated ion channel polypeptide is expressed in a cell. In someembodiments, the cell is an excitable cell. According to another aspectof the invention, methods of altering ion conductivity of a membrane areprovided, the methods including expressing in a host membrane at leastone of a light-activated ion channel polypeptide comprising an aminoacid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, or afunctional variant thereof and contacting the at least one of theexpressed light-activated ion channel polypeptides with a light thatactivates at least one of the light-activated ion channels and altersthe ion conductivity of the host membrane. In some embodiments, the hostmembrane is in a cell.

According to another aspect of the invention, methods of assessing theeffect of a candidate compound on ion conductivity of a membrane areprovided, the methods including: (a) contacting a test membranecomprising the light-activated ion channel polypeptide comprising theamino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or afunctional variant thereof with light under conditions suitable foraltering ion conductivity of the membrane; (b) contacting the testmembrane with a candidate compound; and (c) identifying the presence orabsence of a change in ion conductivity of the membrane contacted withthe light and the candidate compound compared to ion conductivity in acontrol cell contacted with the light and not contacted with thecandidate compound; wherein a change in the ion conductivity in the testmembrane compared to the control indicates an effect of the candidatecompound on the ion conductivity of the test membrane. In someembodiments, the test membrane is in a test cell. In certainembodiments, altering the ion conductivity of the test membranedepolarizes the test cell.

According to another aspect of the invention, a cell is provided thatcomprises an embodiment of any of the aforementioned vectors. Accordingto another aspect of the invention, a cell is provided that comprises anembodiment of any of the aforementioned aspects of light-activated ionchannel polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B provides sequences and a schematic diagram of amino acids.FIG. 1A shows aligned amino acid sequences, set forth herein as SEQ IDNOs: 19-23. SEQ ID NO: 19 is a fragment of full-length ChR1 polypeptide,SEQ ID NO: 20 is a fragment of full-length ChR2 polypeptide; SEQ ID NO:21 is a fragment of full-length VChR1 polypeptide; SEQ ID NO: 22 is afragment of full-length VChR2 polypeptide; and SEQ ID NO: 23 is afragment of full-length BR polypeptide. FIG. 1B shows schematic drawingsof amino acids.

FIG. 2A-C provides schematic diagrams of certain materials used toprepare embodiments of Chronos single and double slow mutants. FIG. 2Ashows PN3-Chronos C145S/D173A-mCherry construct. FIG. 2B showsPN3-Chronos C145S-mCherry construct. FIG. 2C shows PN3-Chronos-mCherryconstruct.

FIG. 3A-B shows recorded traces generated by contacting expressedlight-activated ion channel polypeptides with illumination 470 nm at 5ms. FIG. 3A shows normalized current traces, 20 s long. FIG. 3A showsgenerated photocurrent amplitudes of Chronos and two embodiments of slowkinetics mutants of the invention: a Chronos C145S single mutantpolypeptide and a Chronos C145S/D173A double mutant polypeptide. Toptrace (darkest) is that of the Chronos polypeptide. Of the threepolypeptides, the Chronos trace shows the steepest, most rapid rise tozero after activation. The middle trace provides results of theactivation of a Chronos C145S/D173A double mutant polypeptide that isfollowed by a slower initial rise than either of the other two traces.The trace generated using the activation conditions on an expressedChronos C145S single mutant polypeptide showed an initial sharp upwardslope followed by a less-sharp rise. An examination of photo-currents inclose-up (FIG. 3B) showed that Chronos C145S/D173A double mutant had aslower photo-current activation than both Chronos and Chronos C145Ssingle mutant. In FIGS. 3A and B show results with Chronos-mCherry(control), Chronos-C145S-mCherry (single mutant); andChronos-C145S/D173A-mCherry (double mutant). In FIGS. 3A and B, thevertical axis is normalized photo-current; the horizontal axis is timein milliseconds; the solid box above traces indicates illuminationperiod.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is amino acid sequence of a slow Chronos mutant polypeptide(single mutant—includes C145 S).

METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTSPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.

SEQ ID NO: 2 is a mammalian-codon optimized DNA sequence encoding SEQ IDNO: 1, which is a slow Chronos mutant polypeptide.

atggaaacagccgccacaatgacccacgcctttatctcagccgtgcctagcgccgaagccacaattagaggcctgctgagcgccgcagcagtggtgacaccagcagcagacgctcacggagaaacctctaacgccacaacagccggagccgatcacggttgcttcccccacatcaaccacggaaccgagctgcagcacaagatcgcagtgggactccagtggttcaccgtgatcgtggctatcgtgcagctcatcttctacggttggcacagcttcaaggccacaaccggctgggaggaggtctacgtctgcgtgatcgagctcgtcaagtgcttcatcgagctgttccacgaggtcgacagcccagccacagtgtaccagaccaacggaggagccgtgatttggctgcggtacagcatgtggctcctgactagccccgtgatcctgatccacctgagcaacctgaccggactgcacgaagagtacagcaagcggaccatgaccatcctggtgaccgacatcggcaacatcgtgtgggggatcacagccgcctttacaaagggccccctgaagatcctgttcttcatgatcggcctgttctacggcgtgacttgcttcttccagatcgccaaggtgtatatcgagagctaccacaccctgcccaaaggcgtctgccggaagatttgcaagatcatggcctacgtcttcttctgctcttggctgatgttccccgtgatgttcatcgccggacacgagggactgggcctgatcacaccttacaccagcggaatcggccacctgatcctggatctgatcagcaagaacacttggggcttcctgggccaccacctgagagtgaagatccacgagcacatcctgatccacggcgacatccggaagacaaccaccatcaacgtggccggcgagaacatggagatcgagaccttcgtcgacgaggaggaggagggaggagtg.

SEQ ID NO: 3 is amino acid sequence of a slow Chronos mutant polypeptide(double mutant includes C145S and D173A).

METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTSPVILIHLSNLTGLHEEYSKRTMTILVTAIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.

SEQ ID NO: 4 is a mammalian-codon optimized DNA sequence encoding SEQ IDNO: 3, which is a slow Chronos mutant polypeptide.

atggaaacagccgccacaatgacccacgcctttatctcagccgtgcctagcgccgaagccacaattagaggcctgctgagcgccgcagcagtggtgacaccagcagcagacgctcacggagaaacctctaacgccacaacagccggagccgatcacggttgcttcccccacatcaaccacggaaccgagctgcagcacaagatcgcagtgggactccagtggttcaccgtgatcgtggctatcgtgcagctcatcttctacggttggcacagcttcaaggccacaaccggctgggaggaggtctacgtctgcgtgatcgagctcgtcaagtgcttcatcgagctgttccacgaggtcgacagcccagccacagtgtaccagaccaacggaggagccgtgatttggctgcggtacagcatgtggctcctgactagccccgtgatcctgatccacctgagcaacctgaccggactgcacgaagagtacagcaagcggaccatgaccatcctggtgaccgcaatcggcaacatcgtgtgggggatcacagccgcctttacaaagggccccctgaagatcctgttcttcatgatcggcctgttctacggcgtgacttgcttcttccagatcgccaaggtgtatatcgagagctaccacaccctgcccaaaggcgtctgccggaagatttgcaagatcatggcctacgtcttcttctgctcttggctgatgttccccgtgatgttcatcgccggacacgagggactgggcctgatcacaccttacaccagcggaatcggccacctgatcctggatctgatcagcaagaacacttggggcttcctgggccaccacctgagagtgaagatccacgagcacatcctgatccacggcgacatccggaagacaaccaccatcaacgtggccggcgagaacatggagatcgagaccttcgtcgacgaggaggaggagggaggagtg.

SEQ ID NO: 5 is transmembrane region of SEQ ID Nos: 1 and 3, thatincludes residues corresponding to amino acids 61-295 of SEQ ID NO: 1,which is a slow Chronos mutant polypeptide.

GTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIH.

SEQ ID NO: 6 is amino acid sequence of Chronos (ChR90) polypeptide (SeePCT Publication No. WO 2013/071231)

METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.

SEQ ID NO: 7 is a mammalian-codon optimized DNA sequence encoding ChR90light-activated ion channel polypeptide (See PCT Publication No. WO2013/071231)

atggaaacagccgccacaatgacccacgcctttatctcagccgtgcctagcgccgaagccacaattagaggcctgctgagcgccgcagcagtggtgacaccagcagcagacgctcacggagaaacctctaacgccacaacagccggagccgatcacggttgcttcccccacatcaaccacggaaccgagctgcagcacaagatcgcagtgggactccagtggttcaccgtgatcgtggctatcgtgcagctcatcttctacggttggcacagcttcaaggccacaaccggctgggaggaggtctacgtctgcgtgatcgagctcgtcaagtgcttcatcgagctgttccacgaggtcgacagcccagccacagtgtaccagaccaacggaggagccgtgatttggctgcggtacagcatgtggctcctgacttgccccgtgatcctgatccacctgagcaacctgaccggactgcacgaagagtacagcaagcggaccatgaccatcctggtgaccgacatcggcaacatcgtgtgggggatcacagccgcctttacaaagggccccctgaagatcctgttcttcatgatcggcctgttctacggcgtgacttgcttcttccagatcgccaaggtgtatatcgagagctaccacaccctgcccaaaggcgtctgccggaagatttgcaagatcatggcctacgtcttcttctgctcttggctgatgttccccgtgatgttcatcgccggacacgagggactgggcctgatcacaccttacaccagcggaatcggccacctgatcctggatctgatcagcaagaacacttggggcttcctgggccaccacctgagagtgaagatccacgagcacatcctgatccacggcgacatccggaagacaaccaccatcaacgtggccggcgagaacatggagatcgagaccttcgtcgacgaggaggaggagggaggagtg.

SEQ ID NO: 8 is the mammalian codon-optimized DNA sequence that encodesthe wild-type Channelrhodopsin-2, (see: Boyden, E. et al., NatureNeuroscience 8, 1263-1268 (2005) and Nagel, G., et al. PNAS Nov. 25,2003 vol. 100 no. 24 13940-13945), also referred to herein as ChR2:

atggactatggcggcgctttgtctgccgtcggacgcgaacttttgttcgttactaatcctgtggtggtgaacgggtccgtcctggtccctgaggatcaatgttactgtgccggatggattgaatctcgcggcacgaacggcgctcagaccgcgtcaaatgtcctgcagtggcttgcagcaggattcagcattttgctgctgatgttctatgcctaccaaacctggaaatctacatgcggctgggaggagatctatgtgtgcgccattgaaatggttaaggtgattctcgagttcttttttgagtttaagaatccctctatgctctaccttgccacaggacaccgggtgcagtggctgcgctatgcagagtggctgctcacttgtcctgtcatccttatccacctgagcaacctcaccggcctgagcaacgactacagcaggagaaccatgggactccttgtctcagacatcgggactatcgtgtggggggctaccagcgccatggcaaccggctatgttaaagtcatcttcttttgtcttggattgtgctatggcgcgaacacattttttcacgccgccaaagcatatatcgagggttatcatactgtgccaaagggtcggtgccgccaggtcgtgaccggcatggcatggctgtttttcgtgagctggggtatgttcccaattctcttcattttggggcccgaaggttttggcgtcctgagcgtctatggctccaccgtaggtcacacgattattgatctgatgagtaaaaattgttgggggttgttgggacactacctgcgcgtcctgatccacgagcacatattgattcacggagatatccgcaaaaccaccaaactgaacatcggcggaacggagatcgaggtcgagactctcgtcgaagacgaagccgaggccggagccgtg.

SEQ ID NO: 9 is the amino acid sequence of the wild-typeChannelrhodopsin-2, (see: Boyden, E. et al., Nature Neuroscience 8,1263-1268 (2005) and Nagel, G., et al. PNAS Nov. 25, 2003 vol. 100 no.24 13940-13945), also referred to herein as ChR2:

MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLV EDEAEAGAV.

SEQ ID NO: 10 is amino acid sequence of Chrimson polypeptide:

MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDE DTV.

SEQ ID NO: 11 is amino acid sequence of a slow Chrimson polypeptide withC170S substitution:

MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSSPVILIKLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDE DTV.

SEQ ID NO: 12 is amino acid sequence of a slow Chrimson polypeptide withC170S and C198A substitutions:

MAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSSPVILIKLSNLSGLKNDYSKRTMGLIVSAVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDE DTV.

SEQ ID NO: 13 is amino acid sequence of CoChR polypeptide:

MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTCPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV.

SEQ ID NO: 14 is amino acid sequence of a slow CoChR polypeptide withC108S substitution:

MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTSPVILIHLSNLTGLKDDYSKRTMRLLVSDVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV.

SEQ ID NO: 15 is amino acid sequence of a slow CoChR polypeptide withC108S and D136A substitutions:

MLGNGSAIVPIDQCFCLAWTDSLGSDTEQLVANILQWFAFGFSILILMFYAYQTWRATCGWEEVYVCCVELTKVIIEFFHEFDDPSMLYLANGHRVQWLRYAEWLLTSPVILIHLSNLTGLKDDYSKRTMRLLVSAVGTIVWGATSAMSTGYVKVIFFVLGCIYGANTFFHAAKVYIESYHVVPKGRPRTVVRIMAWLFFLSWGMFPVLFVVGPEGFDAISVYGSTIGHTIIDLMSKNCWGLLGHYLRVLIHQHIIIYGDIRKKTKINVAGEEMEVETMVDQEDEETV.

SEQ ID NO: 16 is amino acid sequence of CsChR polypeptide:

MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGAVVPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTCPVILIHLSNLTGMKNDYNKRTMALLVSDVGCIVWGTTAALSTDFVKIIFFFLGLLYGFYTFYAAAKIYIEAYHTVPKGICRQLVRLQAYDFFFTWSMFPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHEHILVHGNITKKTKVNVAGDMVELDTYVDQDEEHDEG.

SEQ ID NO: 17 is amino acid sequence of a slow CsChR polypeptide withC165S substitution:

MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGAVVPEDHFVCGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTSPVILIHLSNLTGMKNDYNKRTMALLVSDVGCIVWGTTAALSTDFVKIIFFFLGLLYGFYTFYAAAKIYIEAYHTVPKGICRQLVRLQAYDFFFTWSMFPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHEHILVHGNITKKTKVNVAGDMVELDTYVDQDEEHDEG.

SEQ ID NO: 18 is amino acid sequence of a slow CsChR polypeptide withC165S and D193A substitutions:

MSRLVAASWLLALLLCGITSTTTASSAPAASSTDGTAAAAVSHYAMNGFDELAKGAVVPEDHFVGPADKCYCSAWLHSHGSKEEKTAFTVMQWIVFAVCIISLLFYAYQTWRATCGWEEVYVTIIELVHVCFGLWHEVDSPCTLYLSTGNMVLWLRYAEWLLTSPVILIHLSNLTGMKNDYNKRTMALLVSAVGCIVWGTTAALSTDFVKIIFFFLGLLYGFYTFYAAAKIYIEAYHTVPKGICRQLVRLQAYDFFFTWSMEPILFMVGPEGFGKITAYSSGIAHEVCDLLSKNLWGLMGHFIRVKIHEHILVHGNITKKTKVNVAGDMVELDTYVDQDEEHDEG.

SEQ ID NO: 19 is amino acid sequence of a fragment of a full-length ChR1polypeptide:

NKTVWLRYAEWLLTCPVILIHLS.

SEQ ID NO: 20 is amino acid sequence of a fragment of a full-length ChR2polypeptide:

HRVQWLRYAEWLLTCPVILIHLS.

SEQ ID NO: 21 is amino acid sequence of a fragment of a full-lengthVChR1 polypeptide:

NGVVWMRYGEWLLTCPVLLIHLS.

SEQ ID NO: 22 is amino acid sequence of a fragment of a full-lengthVChR2 polypeptide:

NRVLWLRYGEWLLTCPVILIHLS.

SEQ ID NO: 23 is amino acid sequence of a fragment of a full-length BRpolypeptide:

NPIYWARYADWLFTTPLTLLDLA.

SEQ ID NO: 24 is the DNA sequence of the ER export sequence (alsoreferred to herein as

ttctgctacgagaatgaagtg.

SEQ ID NO: 25 is the amino acid sequence of the ER export sequenceencoded by SEQ ID NO: 24 and also referred to herein as “ER2”:

FCYENEV.

SEQ ID NO: 26 is the DNA sequence of KGC, which is a C terminal exportsequence from the potassium channel Kir2.1:

aaatccagaattacttctgaaggggagtatatccctctggatcaaataga catcaatgtt.

SEQ ID NO: 27 is the amino acid sequence of KGC encoded by SEQ ID NO:26, which is a C terminal export sequence from the potassium channelKir2.1:

KSRITSEGEYIPLDQIDINV.

SEQ ID NO: 28 is the DNA sequence of SS, which is a signal peptide thatis destined towards the secretory pathway:

atggtcccgtgcacgctgctcctgctgttggcagccgccctggctccgac tcagacgcgggcc.

SEQ ID NO: 29 is the amino acid sequence of SS encoded by SEQ ID NO: 28:

MVPCTLLLLLAAALAPTQTRA.

SEQ ID NO: 30 is nucleic acid sequence of synapsin promoter, alsoreferred to herein as “syn”:

ctagactgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcggggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattccccaaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcactgccagcttcagcaccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctcagcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttggtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggcacgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctcagtctgcggtgggcagcggaggagtcgtgtcgtgcctgagagcgcagtcgaga.

SEQ ID NO: 31 is nucleic acid sequence of a hemagglutinin:

tacccatacgatgttccagattacgct.

SEQ ID NO: 32 is amino acid sequence of the hemagglutinin polypeptideencoded by SEQ ID NO:

YPYDVPDYA.

DETAILED DESCRIPTION

The invention in some aspects relates to the expression in cells ofstimulus-driven ion channel polypeptides that can be activated bycontact with one or more pulses of light, which results in strongdepolarization of the cell. Embodiments of the invention include opsinpolypeptides comprising sequences that have been identified anddetermined to result in altered channel kinetics of the opsin molecules.Light-activated ion channel polypeptides of the invention, also referredto herein as “slow mutant” polypeptides and “slow mutant light activatedion channel” polypeptides can be expressed in specific cells, tissues,and/or organisms and used to control cells in vivo, ex vivo, and invitro in response to pulses of light of a suitable wavelength. Specificamino acid substitutions and combinations of substitutions have now beenidentified that when present in a sequence of a light-activated ionchannel polypeptide expressed in a membrane and contacted with an“activating” light, alter the response time and/or length of“open-state” time of the substituted light-activated ion channelpolypeptide compared to the same light-activated ion channel polypeptidewithout the one or substitutions.

Slow mutant polypeptide sequences have now been identified that arederived from parent light-activated ion channel amino acid sequences,which are also referred to herein as “non-slow mutant” parentpolypeptides or “non-slow mutant ion channel” parent polypeptides.Non-limiting examples of non-slow mutant parent polypeptides are:Chronos, Chrimson, CsChR, CoChR polypeptides. When a slow mutantpolypeptide of the invention is activated with light, its ion channelremains open for a longer period of time than does the ion channel ofthe slow mutant's light-activated parent polypeptide under similaractivation conditions. In some embodiments, the length of time the slowmutant polypeptide of the invention remains open is statisticallysignificant compared to the shorter open time of a controllight-activated ion channel polypeptide. In a non-limiting example, theion channel of a slow mutant light-activated polypeptide that has aChronos polypeptide parent, maintains an open time that is longer thanthe period of ion channel open time of its parent, when each isactivated under similar conditions. Although slow mutant polypeptides ofthe invention have been determined to maintain a longer open time thantheir parents when activated, slow mutant polypeptides share certaincharacteristics with their respective parents. For example, though notintended to be limiting, slow mutants and their parent molecules arestrongly activated by sufficient contact with a suitable wavelengthlight, can be delivered to cells and subjects and expressed in cellmembranes using similar delivery and administration means, andactivation opens the ion channel of the polypeptide. In some aspects ofthe invention, the parent molecule is a Chronos, Chrimson, CsChR, CoChRpolypeptide or functional variant thereof, or its encoding nucleic acid.Methods to activate light-activated ion channel polypeptides such asChronos, Chrimson, CsChR, CoChR, and variants thereof, are known in theart and include knowledge and use of variables such as, but not limitedto: wavelength of contacting light, pulse length of contacting light,light pulse frequency, pattern of contact with the light, etc. In someembodiments of the invention, these and other art-known methods can beused to activate a corresponding slow mutant polypeptide of theinvention and to open the channel of the slow mutant polypeptide.

Optogenetic tools such as light-activated ion channel polypeptides areused in many fields, including but not limited to, in research and intherapeutic preclinical and clinical applications. However, due to thelevel of expression of light-activated polypeptides that may be achievedin neurons, contact with sufficient light is necessary to activate theexpressed polypeptides. Light levels needed to activate previously knownlight-activated ion channel polypeptides may be greater than 1 mW mm⁻²intensity and must be applied to tissue in which light-activatedpolypeptides are expressed over the time period in which depolarizationis required, which has been difficult in long time-scale experiments.The slow mutant light-activated channel polypeptides of the inventiondiffer from prior light-activated ion channel polypeptides in that whena slow mutant polypeptide of the invention is contacted with light andits channel opens, the channel remains open for a period of time thatpermits less light to be used to maintain sufficient activation ascompared to the light required for use of non-slow mutant ion channelpolypeptides. Thus, slow mutant polypeptides of the invention can beused in experiments, treatments, and other methods for which previouslyknown non-slow mutant light activated ion channel polypeptides have beenused, but the use of slow mutant polypeptides permit activation and opentimes to be maintained with less light exposure and contact, therebyreducing negative effects associated with longer light exposure.

Embodiments of light-activated ion channel polypeptides of theinvention, as compared to prior light-activated polypeptides have anincreased responsiveness to lower levels of activating light. Becausephotocurrent amplitudes at a given light intensity are set, at least inpart, by a balance between recruitment of new open states and transitionto the closed state, the extended length “open time” of slow mutantpolypeptide ion channels of the invention may result in increasedphotocurrent, under lower light exposure conditions. For example,contact of a plurality of slow mutant light-activated ion channelpolypeptides of the invention expressed in cells with a suitableactivating light can result in increased accumulations of the channelsin the plurality of polypeptides that are in the open state, whichresults in effectively increased responsiveness at lower light levelscompared to previously known light-activated ion channel polypeptides.

The invention, in part, includes slow mutant light-activated ion channelpolypeptides in which amino acid substitutions have been made in theamino acid sequence of a parent light activated ion channel polypeptidethat includes in the structural helix known as helix 3 of the seventransmembrane helices in the polypeptide. Amino acid substitutions inslow mutants of the invention result in reduced interactions between thealtered polypeptide region and the all-trans retinal Schiff base (RSB)chromophore. Slow mutants of the invention include changes in the aminoacid sequence that interfere with the RSB and can be used in methodssuch as, but not limited to: color tuning; accumulating conductingstates of the channels in a cell, membrane, and/or organism; andaltering ion channel kinetics—for example, determining the duration ofthe open state of the ion channel following its activation. Thischaracteristic of embodiments of light-activated ion channels of theinvention is also referred to herein as: “increasing accumulations ofthe open state”, maintaining “open-state”, longer “open time”, andincreased “open-time” of the ion channel.

Slow mutant ion channel polypeptides, like their parent light-activatedion channel polypeptides, can be expressed in specific cells, tissues,and/or organisms and used to control cells in vivo, ex vivo, and invitro in response to pulses of light of a suitable activatingwavelength. Slow mutant polypeptides have now been identified thatcomprise a single amino acid substitution or a double amino acidsubstitution in a parent light-activated ion channel polypeptidesequence. Certain slow mutant polypeptides of the invention are derivedfrom parent polypeptides such as Chronos, Chrimson, CsChR, CoChR, andfunctional variants of each of each thereof and include one or twospecific amino acid substitutions to the parent sequences that have nowbeen identified as resulting in an extended channel open time afteractivation, as compared to the open time of the channel of the parentpolypeptide under similar conditions.

Certain embodiments of slow mutant polypeptides of the invention differfrom their parent polypeptides in that following light activation a slowmutant maintains the open state for a statistically significant longerperiod of time than the time period of the open state of its parentpolypeptide under the same activation conditions. In some aspects of theinvention, the parent is a Chronos, Chrimson, CsChR, or CoChRpolypeptide, polynucleotide, or a functional variant of a Chronos,Chrimson, CsChR, or CoChR polypeptide or its encoding polynucleotide.

A non-limiting example of a slow mutant molecule of the invention is aslow mutant Chronos polypeptide or polynucleotide, for example a slowmutant polypeptide comprising the amino acid sequence of the Chronospolypeptide sequence set forth herein as SEQ ID NO: 6, that includes oneor more amino acid substitutions that result in characteristics of aslow mutant light-activated ion channel polypeptide. The slow mutant ofa Chronos polypeptide or its encoding nucleic acid sequence is describedherein as having the Chronos polypeptide or encoding polynucleotide,respectively as its “parent” molecule. A slow mutant polypeptide of theinvention may comprise the amino acid of its parent polypeptide thatincludes one or more amino acid substitutions. In some embodiments theslow mutant of the invention comprises its parent Chronos amino acidsequence with the single amino acid substitution or double amino acidsubstitution, as set forth as SEQ ID NO: 1 and SEQ ID NO: 3,respectively. It will be understood that a functional variant of SEQ IDNO: 6 may also be a parent molecule for a slow mutant of the inventionand that a slow mutant polypeptide of the invention for which afunctional variant of a light-activated ion channel polypeptide such asChronos, Chrimson, ScChR, or CoChR is the parent polypeptide maycomprise the amino acid sequence of the functional variant with thesingle amino acid substitution or double amino acid substitutions thatcorrespond to the substitutions shown in Table 1.

TABLE 1 Identification of substituted residues in positionscorresponding to parent amino acid sequence Parent Name and SingleSubstitution Double Substitutions SEQ ID NO in Slow Mutant in SlowMutant Chronos C145S C145S and D173A (SEQ ID NO: 6) Chrimson C170S C170Sand C198A (SEQ ID NO: 10) CoChR C108S C108S and D136A (SEQ ID NO: 13)CsChR C165S C165S and D193A (SEQ ID NO: 16)

Illumination and Activation

Slow mutant molecules of the invention, include, but are not limited toslow mutant Chronos, slow mutant Chrimson, slow mutant CoChR, slowmutant CsChR, and functional variants thereof [see Klapoetke et al.(2014) Nature Methods 11(3), 338-346; and Yizhar, O. et al. (2011)Neuron Vol. 71:9-34; the content of each of which is incorporated byreference herein in its entirety.] Methods to prepare and expresspreviously known light-activated ion channel molecules can be used inconjunction with the slow mutant molecules described herein. Slow mutantpolypeptides of the invention can be used in art-known methods such as,but not limited to: compound screening, altering cell voltage and/orelectrical activity in cells, and therapeutic methods, which have beendescribed in conjunction with previously known light-activated ionchannel molecules.

Slow mutant polypeptides of the invention can be expressed in fusionproteins and used in optogenetic methods and compositions. Embodimentsof methods of the invention include expressing a slow mutant polypeptideof the invention in a cell and contacting the polypeptide with lightsuitable to activate the polypeptide and open the slow mutantpolypeptide channel. Methods to prepare and express a light-activatedion channel polypeptide in a cell and/or in a subject are well known inthe art, as are methods to select and apply a suitable wavelength oflight to the cell in which the light-activated ion channel is expressedunder suitable conditions to activate the expressed ion channelpolypeptide in the cell.

Specific ranges of wavelengths of light that in some embodiments of theinvention are useful to activate ion channels of the invention areprovided and described herein. It will be understood that a light ofappropriate wavelength for activation and will have a power andintensity appropriate for activation. It is well known in the art thatlight pulse duration, intensity, and power are parameters that can bealtered when activating a channel with light. Thus, one skilled in theart will be able to adjust power, intensity appropriately when using awavelength taught herein or known in the art to activate alight-activated ion channel of the invention. A dose light that contactsa light-activated ion channel of the invention may be determined basedon the wavelength, pulse length, and power of the light that contactsthe light-activated ion channel. Thus, as a non-limiting example, a dosemay have a wavelength of 550 nm, a 4 ms pulse length, and a 0.5 mW/mm²power and another light dose may have a wavelength of 550 nm, a 3 mspulse length and a 0.5 mW/mm² power. Those skilled in the art willunderstand methods to select a dose of light by independently selectinga wavelength, a pulse length, and a power for the light with which alight-activated ion channel of the invention is contacted.

In some embodiments of the invention, wavelength and pulse length may beheld steady, and power incrementally increased to examine activationparameters of a light-activated ion channel of the invention. Similarly,in certain embodiments of the invention may include incrementalwavelength increases while pulse length and power are held steady; orincremental pulse length increases while wavelength and power are heldsteady. In some embodiments of the invention two or more of wavelength,pulse length, and power of a light may be incrementally altered toexamine the effect on activation of a light-activating ion channel ofthe invention. It will be understood that illumination parameters foractivating a parent Chronos, Chrimson, CoChR, or CsChR light-activatedion channel polypeptide that open the polypeptide channel can be used insome embodiments of the invention to activate a slow mutant polypeptideof the invention that is a child of the Chronos, Chrimson, ChChR, orCsChR parent polypeptide, respectively.

Methods of adjusting illumination variables for activatinglight-activated ion channel polypeptides are well-known in the art andmay be applied to activate slow mutant polypeptides of the invention.One example of a benefit of using a slow mutant polypeptide of theinvention is the ability to “tune” the polypeptide's response (forexample, opening, rate of opening, open-time, etc.) using appropriateillumination variables (e.g., wavelength, intensity, duration, etc.),which also referred to herein as dose, to activate the channel. Methodsof adjusting illumination variables are well known in the art andrepresentative methods can be found in publications such as: Lin, J., etal., Biophys. J. 2009 Mar. 4; 96(5):1803-14; Wang, H., et al., 2007 ProcNatl Acad Sci USA. 2007 May 8; 104(19):8143-8. Epub 2007 May 1, each ofwhich is incorporated herein by reference in its entirety. It ispossible to utilize a narrow range of one or more illumination variablesto activate a slow mutant polypeptide of the invention.

Light-Activated Ion Channel Molecules

A slow mutant polypeptide of the invention can be expressed in a cellmembrane and comprises an ion channel that opens upon activation of theslow mutant polypeptide by contact with light under suitable conditions.An ion channel is an integral membrane protein that forms a pore througha membrane and assist in establishing and modulating the small voltagegradient that exists across the plasma membrane of all cells and arealso found in subcellular membranes of organelles such as theendoplasmic reticulum (ER), mitochondria, etc. When a light-activatedion channel of the invention is activated by contacting the cell withappropriate light, the pore opens and permits conductance of ions suchas sodium, potassium, calcium, etc. through the pore.

Slow mutant polypeptides of the invention permit ion conductance anddepolarization when contacted under suitable conditions with anappropriate wavelength of light. As will be understood by those in theart, the term “depolarized” used in the context of cells means an upwardchange in the cell voltage. For example, in an excitable cell at abaseline voltage of about −65 mV, a positive change in voltage, e.g., upto 5, 10, 15, 20, 30, 40, or more millivolts (mV) is a depolarization ofthat cell. When the change in voltage is sufficient to reach the cell'sspike initiation voltage threshold an action potential (e.g. a spike)results. In some embodiments of the invention, activation of the slowmutant polypeptides expressed in a cell membrane results in the voltageof the cell becoming less negative and rising by at least about 20, 30,40, 50, 60, 70, 80, 90, 100 mV (depending on the cell type) thus,depolarizing the cell. As used herein, the term “activate” when used inreference to a slow mutant polypeptide of the invention, means to openthe channel making it permissive to ion conduction and passage throughthe channel.

It has been identified that activating a plurality of at least one slowmutant polypeptide of the invention expressed in a cell or plurality ofcells, results in a channel open-time that permits less illumination tobe used to activate the channels, as compared to non-slow mutantpolypeptides under similar conditions. The channels of activated slowmutant polypeptides remain open for a longer period of time thanchannels of the slow mutants' parent polypeptides when they areactivated under similar conditions.

In some embodiments of the invention, light-activated channels may beused to modify the transmembrane potential (and/or ionic composition) ofcells (and/or their sub-cellular regions, and their local environment).For example, the use of inwardly rectifying cationic channels willdepolarize cells by moving positively charged ions from theextracellular environment to the cytoplasm. Under certain conditions,their use can decrease the intracellular pH (and/or cationconcentration) or increase the extracellular pH (and/or cationconcentration). In some embodiments, the presence of light-activated ionchannels in one, two, three, or more (e.g. a plurality) of cells in atissue or organism, can result in depolarization of the single cell orthe plurality of cells by contacting the light-activated ion channelswith light of suitable wavelength.

The invention, in part, also includes polynucleotides comprising nucleicacid sequences that encode slow mutant polypeptides and functionalvariants thereof of the invention as well as vectors and constructs thatcomprise such nucleic acid sequences. In some embodiments the inventionincludes expression in cells, tissues, and subjects of slow mutantpolypeptides encoded by the nucleic acid sequences. In certainembodiments, the invention comprises methods for preparing and includedin vectors genes that encode slow mutant polypeptides of the invention.The vectors may be delivered into, also referred to herein as:“administered to” a cell and/or subject and the encoded slow mutantpolypeptide expressed in the cell and/or subject.

The invention, in part, includes isolated nucleic acids comprisingsequences that encode light-activated ion channels of the invention aswell as vectors and constructs that comprise such nucleic acidsequences. Light-activated ion channel polypeptides of the invention maybe part of fusion proteins. Thus, a fusion protein may comprise alight-activated ion channel of the invention and may be used in methodsof the invention. Also encompassed by embodiments of the invention aremethods for preparing and using genes that encode slow mutantlight-activated ion polypeptides, including, but not limited to,expressing in cells, tissues, and organisms, one or more slow mutantpolypeptides encoded by the nucleic acid sequences. The terms,“protein,” “polypeptides,” and “peptides” are used interchangeablyherein.

Sequences

The present invention, in part, includes novel light-activated ionchannels polypeptides and methods of their use in cells and subjects.Non-limiting examples of sequences of slow mutant polypeptides of theinvention are set forth herein as SEQ ID NO: 1, 3, 11, 12, 14, 15, 17,and 18. The slow mutant polypeptides of the invention also includefunctional variants of polypeptides set forth as SEQ ID NOs: 1, 3, 11,12, 14, 15, 17, and 18.

A functional variant or modified slow mutant polypeptide of theinvention versus its parent slow mutant polypeptide may comprise (1)substitutions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more aminoacids, (2) insertions and/or deletions of at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more amino acids at one or several positions. It will beunderstood that modification or change in a parent slow mutantpolypeptide amino acid sequence will not include a change in the singleor double substitutions identified in Table 1. Thus, for example, afunctional variant of SEQ ID NO: 1 may include one or moresubstitutions, deletions, and/or insertions to the sequence of SEQ IDNO: 1, but will include a serine in the position that corresponds toamino acid 145 of SEQ ID NO: 1. Similarly, a functional variant of SEQID NO: 12 may include one or more substitutions, deletions, and/orinsertions to the sequence of SEQ ID NO: 12, but will include a serinein the position that corresponds to amino acid 170 and an alanine in theposition that corresponds to amino acid 198 of SEQ ID NO: 12. Selectionand preparation of sequence modifications in slow light-activated ionchannel polypeptides of the invention for preparation and use offunctional variants can be carried out using routine methods. Extensiveinformation on modifications that can be include that will not eliminatefunction of a slow light-activated ion channel polypeptide is availablein the art. [See for example, see: Klapoetke et al, (2014) NatureMethods, March; 11(3):338-346 including supplement, the entire contentof which is incorporated herein by reference.]

A functional variant of a slow mutant polypeptide of the invention mayhave at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, amino acid sequence identity with its parent slow mutantpolypeptide. Thus, a functional variant of SEQ ID NO: 1, 3, 11, 12, 14,15, 17, or 18 has at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%, amino acid sequence identity with its parent slowmutant polypeptide: SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, or 18,respectively. As used herein, the term “identity” refers to the degreeof relatedness between two or more polypeptide sequences, which may bedetermined by the match between the sequences. The percentage isobtained as the percentage of identical amino acids in two or moresequences taking account of gaps and other sequence features. Theidentity between polypeptide sequences can be determined by means ofknown procedures. Algorithms and programs are available and routinelyused by those in the art to determine identity between polypeptidesequences. Non-limiting examples of programs and algorithms includeBLASTP, BLASTN and FASTA (Altschul et al., NCB NLM NIH Bethesda Md.20894; Altschul et al., 1990), Online BLAST programs from the NationalLibrary of Medicine are available, for example, atblast.ncbi.nlm.nih.gov/Blast.cgi.

A non-limiting example of a functional variant of SEQ ID NO: 1 has theamino acid sequence set forth as SEQ ID NO: 1 including serine at theposition corresponding to residue 145 of SEQ ID NO: 1, but includesmodifications comprising one or more of substitutions: A18G, A36G, D51E,I68V, A94G, I113L, I113V, R165K, A210G, and I257V. A non-limitingexample of a functional variant of SEQ ID NO: 3 has the amino acidsequence set forth as SEQ ID NO: 3 including the serine and alanine atpositions corresponding to residues 145 and 173, respectively of SEQ IDNO: 3, but includes modifications comprising one or more ofsubstitutions: A18G, A36G, D51E, I68V, A94G, I113L, I113V, R165K, A210G,and I257V. A non-limiting example of a functional variant of SEQ ID NO:11 has the amino acid sequence set forth as SEQ ID NO: 11 including theserine at the position corresponding to residue 170 of SEQ ID NO: 11,but includes modifications comprising one or more of substitutions: ABG,D29E, D56E, R85K, A101G, A119G, I221L, and I221V. A non-limiting exampleof a functional variant of SEQ ID NO: 12 has the amino acid sequence setforth as SEQ ID NO: 12 including the serine and alanine at positionscorresponding to residues 170 and 198, respectively of SEQ ID NO: 12,but includes modifications comprising one or more of substitutions: ABG,D29E, D56E, R85K, A101G, A119G, I221L, and I221V. A non-limiting exampleof a functional variant of SEQ ID NO: 14 comprises the amino acidsequence set forth as SEQ ID NO: 14 including the serine at the positioncorresponding to residue 108 of SEQ ID NO: 14, but includesmodifications comprising one or more of substitutions: D21E, I44L, I44V,R56K, I75L, I75V, I156L, I156V, and D223E. A non-limiting example of afunctional variant of SEQ ID NO: 15 comprises the amino acid sequenceset forth as SEQ ID NO: 15 including serine and alanine at positionscorresponding to residues 108 and 136, respectively of SEQ ID NO: 15,but includes modifications comprising one or more of substitutions:D21E, I44L, I44V, R56K, I75L, I75V, I156L, I156V, and D223E. Anon-limiting example of a functional variant of SEQ ID NO: 17 comprisesthe amino acid sequence set forth as SEQ ID NO: 17 including the serineat the position corresponding to residue 165 of SEQ ID NO: 17, butincludes modifications comprising one or more of substitutions: A12G,D50E, A88G, A108G, I126L, I126V, R157K, R246K, and A279G. A non-limitingexample of a functional variant of SEQ ID NO: 18 comprises the aminoacid sequence set forth as SEQ ID NO: 18 including the serine andalanine at positions corresponding to residues 165 and 193, respectivelyof SEQ ID NO: 18, but includes sequence modifications comprising one ormore of substitutions: A12G, D50E, A88G, A108G, I126L, I126V, R157K,R246K, and A279G.

TABLE 2 Non-limiting examples of substituted slow light-activated ionchannel polypeptides of the invention, each also referred to herein avariant of its parent sequence. Each of the below examples shows from 1to 5 amino acid substitutions to a parent sequence. AdditionalAdditional Additional Additional Additional Parent Modifica- Modifica-Modifica- Modifica- Modifica- SEQ ID tion tion tion tion tion SEQ IDA18G NO: 1  SEQ ID A18G D51E NO: 1  SEQ ID A18G D51E I113L NO: 1  SEQ IDI113L A210G ON: 1  SEQ ID A36G I113V A210G I257V NO: 1  SEQ ID A18G NO:3  SEQ ID I113L NO: 3  SEQ ID D51E A94G A210G NO: 3  SEQ ID A36G A94GI113V A210G I257V ON: 3  SEQ ID A8G D56E NO: 11 SEQ ID D29E R85K A119GI221V NO: 11 SEQ ID A8G D56E R85K A119G I221L NO: 11 SEQ ID D56E A119GNO: 11 SEQ ID A8G ON: 12 SEQ ID A101G NO: 12 SEQ ID D29E R85K I221V NO:12 SEQ ID A8G D56E A101G A119G I221L NO: 12 SEQ ID D21E NO: 14 SEQ IDI75L I156V ON: 14 SEQ ID I44L R56K I156L D223E NO: 14 SEQ ID D21E I44VR56K I75L I156V NO: 14 SEQ ID D21E NO: 15 SEQ ID I44V I75L I157L D223ENO: 15 SEQ ID D21E I44L I75V I156V D223E NO: 15 SEQ ID I75V D223E ON: 15SEQ ID A12G A88G NO: 17 SEQ ID D50E NO: 17 SEQ ID A12G A108G R157K R246KA279G NO: 17 SEQ ID I126V R157K NO: 17 SEQ ID A12G ON: 18 SEQ ID A88GI126L NO: 18 SEQ ID D50E A88G I126V R157K A279G NO: 18 SEQ ID I126VR246K NO: 18

One skilled in the art will understand that slow mutant light-activatedion channels of the invention can be identified based on sequencesimilarity to a slow mutant polypeptide disclosed and described herein.It will be understood that additional slow mutant polypeptides may beidentified using sequence alignment with one of the slow mutantpolypeptides sequences or functional variants thereof described herein.Sequence identity can be determined using standard techniques known inthe art.

For slow mutant polypeptides and variants thereof of the invention, thepresence of functionality, e.g., activation of a channel by contact withsuitable light, length of open-time of a channel, brightness ofillumination required for activation, etc. can be determined usingmethods described herein, and functional variants of slow mutantpolypeptides of the invention can be used in methods described herein.It is understood that the level of sequence identity with a slow mutantpolypeptide of the invention plus functionality with respect toactivation by suitable light, open-time, and other illuminationvariables can be characteristics used to identify additional slow mutantpolypeptides using standard procedures for sequence alignment,comparisons, assays for channel polypeptide activation, and assays forion channel activity.

Slow mutant light-activated ion channels of the invention aretransmembrane channel polypeptides that use light energy to open,permitting ion conductance through their pore, thus altering thepotential of the membrane in which they are expressed. A non-limitingexample of an ion that can be moved through a pore of the inventionincludes a sodium ion, a potassium ion, a calcium ion, a proton, etc.Routine methods may be used to measure different ion currents forlight-activated ion channels of the invention. Slow mutant polypeptidesof the invention can be activated by sustained light and/or by lightpulses and the ion conductance resulting from activation of a slowmutant polypeptides of the invention can depolarize cells and alter thevoltage in cells and organelles in which they are expressed.

In non-limiting examples of implementations, the invention comprisesmethods for preparing and using genes encoding light-activated ionchannels of the invention that have now been identified. The invention,in part, also includes isolated nucleic acids comprising sequences thatencode light activated ion channels of the invention as well as vectorsand constructs that comprise such nucleic acid sequences. In someembodiments the invention includes expression of polypeptides encoded bythe nucleic acid sequences, in cells, tissues, and organisms.

The slow mutant polypeptides and their encoding nucleic acid sequencesused in aspects and methods of the invention may be “isolated”sequences. As used herein, the term “isolated” used in reference to apolynucleotide, nucleic acid, amino acid sequence, or polypeptide of theinvention, means a polynucleotide sequence, nucleic acid, amino acidsequence or polypeptide that is present in sufficient quantity to permitits identification or use. An isolated nucleic acid or polypeptide ofthe invention is a nucleic acid or polypeptide that is not part of orincluded in a wild-type cell or organism that is a native organism/cellfor its parent molecule. For example, a parent nucleic acid orpolypeptide may be naturally present in a cell or organism of aChloromonas, Chlamydomonas, Stigeoclonium, or other bacterial family,and an isolated slow mutant polypeptide or encoding nucleic acid is aslow mutant molecule that is not located in a cell or organism of abacterial family.

A polypeptide or encoding nucleic acid of a slow mutant of the inventionthat is present in a cell, tissue, and/or organism, etc., is consideredto be in a ‘host’ cell, tissue, and/or organism, respectively.Non-limiting examples of a host membrane, cell, or tissue include:mammalian, non-human primate, vertebrate, invertebrate, fish, reptile,crustaceans, insect, and avian membranes, cells, and tissues.Non-limiting examples of a host organism or subject include: humans,non-human primates, vertebrates, invertebrates, mammals, insects, fish,crustaceans, reptiles, and birds.

Slow Mutant Sequences Including Modified Sequences

A slow mutant molecule of the invention may comprise an amino acidsequence that is modified from its “parent” sequence and the invention,in part, includes functional variants of slow mutant molecules set forthherein. As used herein, a functional variant is a molecule that retainssome or all of one or more functions of its parent sequence, but hasbeen modified from the parent sequence. As used herein the term“modified” or “modification” in reference to a nucleic acid orpolypeptide sequence refers to a change of one, two, three, four, five,six, or more residues in the modified sequence as compared to its parentsequence, which is also referred to herein as the sequence from which itwas derived. For example, a modified polypeptide sequence may beidentical to its parent polypeptide sequence except that it has one,two, three, four, five, or more amino acid substitutions, deletions,insertions, or combinations thereof. In some embodiments of theinvention a modified sequence may include one, two, three, four, or moreamino acid substitutions in a parent polypeptide sequence. In aspects ofan invention, a functional variant of a slow mutant polypeptide or itsencoding polynucleotide has the sequence of its parent slow mutantpolypeptide or its encoding polynucleotide, respectively, but with one,two, three, four, five, or more sequence modifications and stillretaining at some or all of one or more functions of the parentmolecule.

Sequences of slow mutant polypeptides provided herein can be modifiedwith one or more substitutions, deletions, insertions, or othermodifications and such modified light-activated ion channels can betested using methods described herein for characteristics including, butnot limited to: expression, cell localization, activation, open-time,recover, and depolarization in response to contact with light usingmethods disclosed herein. In some embodiments, the invention includesthe use of targeted site-directed mutagenesis at specific amino acidresidues of a slow mutant polypeptide of the invention including but notlimited to residues of one or more of SEQ ID Nos: 1, 3, 11, 12, 14, 15,17, and 18. Specific locations for single mutations can be identifiedand alone, or in combination with two or more additional mutations canbe placed into a slow mutant sequence and tested with respect tocharacteristics such as, but not limited to: their activation,open-time, and photocurrent amplitude. Thus, sequences of slow mutantpolypeptides of the invention can be modified and the resultingpolypeptides tested for various characteristics, and used in methodsdisclosed herein.

Non-limiting examples of modifications that can be included in slowmutant polypeptides of the invention are conservative amino acidsubstitutions, which may produce molecules having functionalcharacteristics similar to those of the molecule from which suchmodifications are made. Conservative amino acid substitutions aresubstitutions that do not result in a significant change in the activityor tertiary structure of a selected polypeptide or protein. Suchsubstitutions typically involve replacing a selected amino acid residuewith a different residue having similar physico-chemical properties. Forexample, substitution of Glu for Asp is considered a conservativesubstitution because both are similarly sized negatively charged aminoacids. Groupings of amino acids by physico-chemical properties are knownto those of skill in the art. The following groups each contain aminoacids that are conservative substitutions for one another: 1) Alanine(A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine(N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine(Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) [see, e.g., Creighton, Proteins, W.H. Freeman, New York(1984)]. Slow mutant polypeptides that include modifications, includingbut not limited to one, two, three, four, or more conservative aminoacid substitutions can be identified and tested for characteristicsincluding, but not limited to: expression, cell localization,activation, open-time, and depolarization and depolarization-effects inresponse to contact with light using methods disclosed herein.

Slow mutant polypeptides of the invention may be shorter or longer thantheir parent light-activated ion channel polypeptide sequences. Thus, aslow mutant polypeptide may be a full-length polypeptide or functionalfragment thereof. In addition, polynucleotides of the invention may beused to obtain additional coding regions, and thus additional slowmutant polypeptide sequences, using techniques known in the art.

In some aspects of the invention, functional variants of a slow mutantpolypeptide sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or 100% identity to a parent slow mutant or to another light-activatedion channel polypeptide sequence disclosed herein, non-limiting examplesof which include CoChR, CsChR, Chrimson, Chronos, etc. Art-knownalignment methods and tools can be used to align substantially similarsequences permitting positional identification of amino acids that maybe modified as described herein to prepare a slow mutant polypeptide ofthe invention or its encoding polynucleotide. Standard sequence analysistools and computer programs, such as those used for alignment, etc. canbe used to identify slow mutant molecules of the invention that shareone or more functional properties with a slow mutant light-activated ionchannel described herein.

Sequence modifications can be in one or more of three classes:substitutions, insertions, or deletions. These modified sequences,(which may also be referred to as variants, or derivatives) may beprepared by site-specific mutagenesis of nucleic acids in the DNAencoding a light-activated ion channel polypeptide, using cassette orPCR mutagenesis or other techniques known in the art, to produce DNAencoding the modified light-activated ion channel polypeptide, andthereafter expressing the DNA in recombinant cell culture, cells, and/orsubjects. Amino acid sequence variants are characterized by thepredetermined nature of the variation, a feature that sets them apartfrom naturally occurring allelic or interspecies variation of thelight-activated ion channels of the invention. Modified slow mutantpolypeptides generally may exhibit the same qualitative biologicalactivity as their parent light-activated ion channel, although variantscan also be selected that have modified characteristics.

A site or region for introducing an amino acid sequence modification maybe predetermined, and the mutation per se need not be predetermined. Forexample, to optimize the performance of a mutation at a given site,random mutagenesis may be conducted at the target codon or region andthe expressed modified light-activated ion channel screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis.

In some aspects of the invention, amino acid substitutions may be singleresidue substitutions; and insertions may be on the order of from 1, 2,3, 4, 5, 6, 7, up to about 20 amino acids, although larger insertionsmay be tolerated. Deletions may range from about 1, 2, 3, 4, 5, 6 7, upto about 20 residues, although in some cases deletions may be larger.Substitutions, deletions, insertions or any combination thereof may beused to arrive at a slow mutant polypeptide of the invention. Generallythese changes are done on a few amino acids to minimize the alterationof the molecule. However, larger changes may be tolerated in certaincircumstances. A modified slow mutant polypeptide of the invention canincorporate unnatural amino acids as well as natural amino acids. Anunnatural amino acid can be included in a light-activated ion channel ofthe invention to enhance a characteristic such as photocurrent,stability, speed, compatibility, open-time, or to lower toxicity, etc.Methods to prepare and functional variants of light-activated moleculesare known and practiced in the art.

Variants of slow mutant polypeptides set forth herein that can be usedin embodiments of methods of the invention may exhibit the samequalitative light-activated ion channel activity as one or more of thesequences set forth herein, such as SEQ ID Nos: 1, 3, 11, 12, 14, 15,17, and 18, but may show some altered characteristics such as alteredphotocurrent, stability, speed, open-time, compatibility, and toxicity,or a combination thereof. For example, the polypeptide can be modifiedsuch that it has an increased open-time, results in increasedphotocurrent and/or has less toxicity than another light-activated ionchannel polypeptide.

Another aspect of the invention provides nucleic acid sequences thatencode slow mutant polypeptides of the invention. It is understood bythose in the art that slow mutant polypeptides of the present inventioncan be coded for by more than one nucleic acid sequence. Each amino acidin the protein is represented by one or more sets of three nucleic acids(codons). Because many amino acids are represented by more than onecodon there is not a unique nucleic acid sequence that codes for a givenprotein. Those in the art will understand how to make and use a nucleicacid sequence that encodes a slow mutant polypeptide of the inventionbased on knowledge of the amino acid sequence of the protein. A nucleicacid sequence that codes for a polypeptide is referred to as the “gene”of that polypeptide. A gene can be RNA, DNA, or other nucleic acid thanwill code for the polypeptide.

It is understood in the art that the codon systems in differentorganisms can be slightly different, and therefore where the expressionof a given protein from a given organism is desired, the nucleic acidsequence can be modified for expression within that organism. Thus, insome embodiments of the invention, a slow mutant polypeptide of theinvention is encoded by a mammalian-codon-optimized nucleic acidsequence, which may in some embodiments be a human-codon optimizednucleic acid sequence. An aspect of the invention provides a nucleicacid sequence that encodes a slow mutant polypeptide of the inventionthat is optimized for expression in a mammalian cell. In someembodiments of the invention, a nucleic acid sequence that encodes aslow mutant polypeptide of the invention includes a nucleic acidsequence optimized for expression in a human cell.

Delivery of Slow Mutant Molecules

Delivery of a slow mutant polypeptide to a cell and/or expression of aslow mutant polypeptide in a cell can be done using art-known deliverymeans. In some embodiments of the invention a slow mutant polypeptide ofthe invention is included in a fusion protein. It is well known in theart how to prepare and utilize fusion proteins that comprise apolypeptide sequence. In certain embodiments of the invention, a fusionprotein can be used to deliver a slow mutant polypeptide into a cell andin some embodiments a fusion protein can be used to target a slow mutantpolypeptide of the invention to specific cells or to specific cells,tissues, or regions in a subject. Targeting and suitable targetingsequences for delivery of a slow mutant polypeptide of the inventioninto a desired cell, tissue or region can be performed using art-knownprocedures.

In some embodiments of the invention, a slow mutant light-activated ionchannel of the invention is genetically introduced into a cellularmembrane, and reagents and methods are provided for genetically targetedexpression of slow mutant polypeptides, including but not limited to:SEQ ID Nos: 1, 3, 11, 12, 14, 15, 17, 18, and functional variants of anythereof. Genetic targeting can be used to deliver one or more slowmutant polypeptides to specific cell types, to specific cell subtypes,to specific spatial regions within an organism, and to sub-cellularregions within a cell. Genetic targeting also relates to the control ofthe amount of a slow mutant polypeptide expressed and the timing of theexpression.

Some embodiments of the invention include a reagent for geneticallytargeted expression of a slow mutant polypeptide, wherein the reagentcomprises a vector that contains the gene for the light-activated ionchannel polypeptide. As used herein, the term “vector” refers to apolynucleotide molecule capable of transporting between differentgenetic environments another nucleic acid sequence to which it has beenoperatively linked. The term “vector” may also refer to a virus ororganism that is capable of transporting the polynucleotide molecule.One type of vector is an episome, i.e., a polynucleotide moleculecapable of extra-chromosomal replication. Some useful vectors are thosecapable of autonomous replication and/or expression of nucleic acidsequences to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors.” Other useful vectors, include, but arenot limited to viruses such as lentiviruses, retroviruses, adenoviruses,and phages. Vectors useful in some methods of the invention cangenetically insert a slow mutant polypeptide including, but not limitedto one set forth herein as: SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, ora functional variant thereof into dividing and non-dividing cells andcan insert slow mutant polypeptides into cells that are in vivo, invitro, or ex vivo cells.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. Non-limitingexamples of suitable expression vectors and methods for their use areprovided herein. Methods suitable to prepare and use expression vectors,polynucleotide sequences, promoters, delivery agents, labeling agents,etc. to express slow mutant polypeptides of the invention are known inthe art.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of a light-activated ionchannel polypeptide in a wide variety of cell types—thus a promoter fora gene that is widely expressed in a variety of cell types, for examplea “housekeeping gene” can be used to express a light-activated ionchannel polypeptide in a variety of cell types. Non-limiting examples ofgeneral promoters are provided elsewhere herein and suitable alternativepromoters are well known in the art. In certain embodiments of theinvention, a promoter may be an inducible promoter.

A plasmid construct may be used in some embodiments of the invention, todeliver and/or express a slow mutant polypeptide of the invention andthe construct may comprise a slow mutant molecule, and one or more of:an AAV-Adeno-associated virus; a trafficking sequence, a signalsequence, an export sequence, a Syn-synapsin promoter, such as but notlimited to SEQ ID NO: 30; an HA-hemagglutinin, such as but not limitedto SEQ ID NO: 31, which encodes SEQ ID NO: 32; an SS-signal sequence,such as but not limited to SEQ ID NO: 28, which encodes SEQ ID NO: 29; atruncated MHC class I antigen; an ER2-Endoplasmic reticulum exportsignal, such as but not limited to SEQ ID NO: 24, which encodes SEQ IDNO: 25; a KGC C terminal export sequence from the potassium channelKir2.1, such as but not limited to SEQ ID NO: 26, which encodes SEQ IDNO: 27. Additional molecules that can be included in constructs for usein methods of the invention, see for example, Kugler, S. et al., GeneTherapy 10, 337-347, (2003); Niman, H. L. et al., Proc. Natl. Acad. Sci.USA 80:4949-4953 (1983); Gradinaru, V. et al., Brain Cell Biol. 36,129-139 (2008); and Chow B. Y. et al., Nature, 463, 98-102(2010), thecontent of each of which is incorporated herein by reference in itsentirety.

As used herein, polypeptide components of a fusion protein, such as, butnot limited to: a slow mutant polypeptide, a targeting polypeptide, asignal polypeptide, a trafficking polypeptide, a detectable labelpolypeptide, may be referred to being “fused” to each other. Forexample, when referring to a slow mutant polypeptide and an targetingpolypeptide that are part of a fusion protein, the slow mutantpolypeptide may be referred to as being “fused” to the targetingpolypeptide. Trafficking polypeptides, export polypeptides, signalpolypeptides, targeting polypeptides are known in the art and can beincluded in a fusion protein to direct the location, (also referred toas: localization), of the expressed polypeptide to a specific cellregion of interest such as a membrane etc.

Compositions of the invention may include a slow mutant molecule and oneor more additional molecules. In some embodiments of the invention, aslow mutant molecule is a polypeptide. In certain embodiments of theinvention, a slow mutant molecule is a polynucleotide with a nucleicacid sequence that encodes a slow mutant polypeptide. In some aspects ofthe invention, a composition comprising a slow mutant molecule of theinvention is a pharmaceutical composition. In some aspects of theinvention the pharmaceutical composition comprises a slow mutantpolypeptide and/or its encoding nucleic acid and a pharmaceuticallyacceptable carrier. Additional components that are optionally includedin a composition include but are not limited to: one or more: vectors,nucleic acid molecules, polypeptides, detectable labels, carriermolecules, targeting molecules, etc.

Functional variants of other components of vectors and/or fusionproteins are also envisioned, for example functional variants of ER2,SS, hemagglutinin, syn promoters, Kir2 sequences and other exportsequences, signal sequences, trafficking sequences etc. that may beinclude in vectors or fusion proteins of the invention.

Component Molecules of Fusion Protein, Vectors, and Compositions

Molecules that may be included in fusion proteins, vectors,compositions, and pharmaceutical compositions of the invention, and canbe expressed in cells in methods of the invention, include but are notlimited to one or more of: slow mutant polypeptides, detectable labelpolypeptides, fluorescent polypeptides, targeting polypeptides,trafficking polypeptides, signal polypeptides, export polypeptides, etc.

Non-limiting examples of detectable label polypeptides that may beincluded in a fusion protein of that also includes a slow mutantpolypeptide of the invention, are: green fluorescent protein (GFP);enhanced green fluorescent protein (EGFP), red fluorescent protein(RFP); yellow fluorescent protein (YFP), tdTomato, mCherry, DsRed, cyanfluorescent protein (CFP); far red fluorescent proteins, etc. In certainaspects of the invention, a fluorescent detectable label polypeptide maybe included, for example for tracking purposes, testing, assays, etc.Numerous fluorescent proteins and their encoding nucleic acid sequencesare known in the art and routine methods can be used to include suchsequences in fusion proteins and vectors, respectively, of theinvention.

Non-limiting of examples of additional amino acid sequences that may beincluded in a fusion protein of the invention are promoter sequences,trafficking sequences, including, but not limited to one or more of thesequences set forth herein as SEQ ID NO: 25, 27, 29, and 32. Additionalamino acid sequences that can be included in a fusion protein of theinvention are known in the art and can be included and used incompositions and methods of the invention using routine methods.

A vector or fusion protein of the invention may also include afunctional variant of a slow mutant molecule of the invention. Forexample a functional variant of SEQ ID NO: 1, 3, parent moleculesincluding but not limited to one or more of: SEQ ID NO: 1-4, 11, 12, 14,15, 17, and 18. A functional variant that is included in a vector or afusion protein of the invention, may have one or more additions,deletions, substitutions, or other modifications to the sequence of itsparent sequence and retains a portion, or all, of the function of itsparent molecule for which the molecule is included in the vector orfusion protein of the invention.

Methods of Use of Slow Mutant Polypeptides

Slow mutant molecules of the invention are well suited for targetingcells and specifically altering voltage-associated cell activities. Insome embodiments of the invention, slow mutant polypeptides of theinvention can be utilized to introduce cations into cells, thusactivating endogenous signaling pathways (such as calcium dependentsignaling), and then drugs are applied that modulate the response of thecell (using a calcium or voltage-sensitive dye). This allows compoundand drug screening using light to activate channels of interest, andusing light to read out the effects of a compound and/or drug on thechannels of interest.

According to certain principles of this invention, slow mutantpolypeptides can be activated to introduce cations into cells, thusactivating endogenous signaling pathways (such as calcium dependentsignaling), and drugs may be applied that modulate the response of thecell (using a calcium or voltage-sensitive dye). Another aspect of theinvention is the use of a slow mutant polypeptide to decrease the pH ofa cell in which it is expressed. Such a technique may be used to treatalkalosis. Another aspect of the invention includes methods of usingslow mutant polypeptides to generate sub-cellular voltage or pHgradients, for example, though not limited to, at synapses and insynaptic vesicles to alter synaptic transmission, and mitochondria toimprove ATP synthesis.

Working operation of prototypes of this invention have been preparedincluding genetically expressing slow mutant polypeptides of theinvention in excitable cells, illuminating the cells with suitablewavelengths of light, and demonstrating rapid depolarization of thecells in response to the light, as well as slow channel closing and slowrelease from depolarization upon cessation of light. Depending on theparticular implementation, methods of the invention allow light controlof cellular functions in vivo, in vitro, or ex vivo. In non-limitingexamples of methods of the invention, slow mutant polypeptides of theinvention have been expressed in cells in human-optimized form allowdepolarization at wavelengths described herein, and have shown extendedopen-times as described versus corresponding non-slow mutantpolypeptides under similar conditions.

Cells and Subjects

A cell used in methods and with sequences of the invention may be anexcitable cell or a non-excitable cell. Cell types in which a slowmutant polypeptide of the invention may be expressed and may be used inmethods of the invention include prokaryotic and eukaryotic cells.Useful cells include but are not limited to mammalian cells. Examples ofcells in which a slow mutant polypeptide of the invention may beexpressed are excitable cells, which include cells able to produce andrespond to electrical signals. Examples of excitable cell types include,but are not limited to neurons, muscles, cardiac cells, and secretorycells (such as pancreatic cells, adrenal medulla cells, pituitary cells,immune system cells, etc.).

Non-limiting examples of cells that may be used in methods of theinvention include: neuronal cells, nervous system cells, cardiac cells,circulatory system cells, immune system cells, visual system cells,auditory system cells, secretory cells, endocrine cells, and musclecells. In some embodiments, a cell in which a slow mutant polypeptide isexpressed and that is used in conjunction with a method of the inventionmay be a healthy normal cell, which is not known to have a disease,disorder or abnormal condition. In some embodiments, a cell used inconjunction with methods and channels of the invention may be anabnormal cell, for example, a cell that has been diagnosed as having adisorder, disease, or condition, including, but not limited to adegenerative cell, a neurological disease-bearing cell, a cell model ofa disease or condition, an injured cell, etc. In some embodiments of theinvention, a cell may be a control cell.

Slow mutant polypeptides of the invention may be expressed in cells inor from culture, cells in solution, cells obtained from subjects, and/orcells in a subject (in vivo cells). Slow mutant polypeptides of theinvention may be expressed and activated in cultured cells, culturedtissues (e.g., brain slice preparations, etc.), and in living subjects,etc. As used herein, the term “subject” may refer to, but is not limitedto: a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat,rodent, fly or another vertebrate or invertebrate organism.

Controls and Candidate Compound Testing

Slow mutant polypeptides of the invention and methods using slow mutantpolypeptides of the invention can be utilized to assess changes incells, tissues, and subjects in which they are expressed. Someembodiments of the invention include use of slow mutant polypeptides ofthe invention to identify effects of candidate compounds on cells,tissues, and subjects. Results of testing a slow mutant polypeptide ofthe invention can be advantageously compared to a control. In someembodiments of the invention one or more slow mutant polypeptides of theinvention, non-limiting examples of which are SEQ ID Nos: 1, 3, 11, 12,14, 15, 17, 18, and functional variants of any thereof, may be expressedin a cell population and used to test the effect of candidate compoundson the cells.

As used herein a control may be a predetermined value, which can take avariety of forms. It can be a single cut-off value, such as a median ormean. It can be established based upon comparative groups, such as cellsor tissues that include the slow mutant polypeptide and are contactedwith light, but are not contacted with the candidate compound and thesame type of cells or tissues that under the same testing condition arecontacted with the candidate compound. Another example of comparativegroups may include cells or tissues that have a disorder or conditionand groups without the disorder or condition. Another comparative groupmay be cells from a group with a family history of a disease orcondition and cells from a group without such a family history. Apredetermined value can be arranged, for example, where a testedpopulation is divided equally (or unequally) into groups based onresults of testing. Those skilled in the art are able to selectappropriate control groups and values for use in comparative methods ofthe invention.

As a non-limiting example of use of a slow mutant polypeptide of theinvention to identify a candidate therapeutic agent or compound, a slowmutant light-activated ion channel of the invention may be expressed inan excitable cell in culture or in a subject and the excitable cell maybe contacted with a light that activates the slow mutant polypeptidechannel and with a candidate therapeutic compound. In one embodiment, atest cell that includes a slow mutant polypeptide of the invention canbe contacted with a light that depolarizes the cell and also contactedwith a candidate compound. The cell, tissue, and/or subject that includethe cell can be monitored for the presence or absence of a change thatoccurs in the test conditions versus the control conditions. Forexample, in a cell, a change may be a change in the depolarization or ina depolarization-mediated cell characteristic in the test cell versus acontrol cell, and a change in depolarization or thedepolarization-mediated cell characteristic in the test cell compared tothe control may indicate that the candidate compound has an effect onthe test cell or tissue that includes the cell. In some embodiments ofthe invention, a depolarization-mediated cell characteristic may be a anaction potential, pH change in a cell, release of a neurotransmitter,etc. and may in some embodiments, include a downstream effect on one ormore additional cells, which occurs due to the depolarization of thecell that includes the slow mutant polypeptide. Art-known methods can besued to assess depolarization and depolarization-mediated cellcharacteristics and changes to the depolarization ordepolarization-mediated cell characteristics upon activation of a slowmutant polypeptide channel of the invention, with or without additionalcontact with a candidate compound.

In some aspects of the invention, a control light-activated ion channelmay be a non-slow mutant light-activated ion channel, which in someembodiments of the invention is a non-slow mutant parent polypeptide.For example, a Chronos polypeptide may serve as a control for a slowmutant polypeptide of the invention for which Chronos is its parent.Similarly, a Chrimson, CoChR, CsChr polypeptide may serve as a controlfor a slow mutant polypeptide of the invention for which a Chrimson,CoChR, CsChr polypeptide, respectively, is the parent.

Candidate-compound identification methods of the invention that areperformed in a subject, may include expressing a slow mutant polypeptidein a subject, contacting the subject with a light under suitableconditions to activate the slow mutant polypeptide and depolarize thecell, and administering to the subject a candidate compound. The subjectis then monitored to determine whether any change occurs that differsfrom a control effect in a subject. Thus, for example, a cell in culturecan be contacted with a light appropriate to activate a slow mutantpolypeptide of the invention in the presence of a candidate compound. Aresult of such contact with the candidate compound can be measured andcompared to a control value as a determination of the presence orabsence of an effect of the candidate compound.

Methods of identifying effects of candidate compounds using slow mutantpolypeptides of the invention may also include additional steps andassays to further characterize an identified change in the cell, tissue,or subject when the cell is contacted with the candidate compound. Insome embodiments, testing in a cell, tissue, or subject can also includeone or more cells that has a slow mutant polypeptide of the invention,and that also has one, two, three, or more additional differentlight-activated ion channels, wherein at least one, two, three, four, ormore of additional types of light-activated ion channels are activatedby contact with light having a different wavelength than used toactivate the slow mutant polypeptide.

In a non-limiting example of a candidate drug identification method ofthe invention, cells that include a slow mutant polypeptide of theinvention are depolarized, thus triggering release of a neurotransmitterfrom the cell, and then drugs are applied that modulate the response ofthe cell to depolarization (determined for example using patch clampingmethods or other suitable art-known means). Such methods enable compoundand drug assays and screening using contact with light under suitableconditions to activate the channels of interest, and using light to readout the effects of the compound or drug on the channels andchannel-containing cells of interest. In some embodiments, slow mutantpolypeptides of the invention can be used in test systems and assays forassessing membrane protein trafficking and physiological function inheterologously expressed systems and the use of methods to activate slowmutant polypeptides to depolarize a cell or plurality of cells.

Expression and Methods of Use in Cells and Subjects

In some embodiments of the invention, a plurality of one or more slowmutant light-activated channel polypeptides can be used to modify thetransmembrane potential (and/or ionic composition) of one or a pluralityof cell. For example, the use of inwardly rectifying cationic channelswill depolarize cells by moving positively charged ions from theextracellular environment to the cytoplasm. Under certain conditions,their use can decrease the intracellular pH (and/or cationconcentration) or increase the extracellular pH (and/or cationconcentration). In some embodiments, the presence of a light-activatedion channel of the invention in one, two, three, or more cells in atissue or organism, can result in depolarization of the single cell orthe plurality of cells by contacting the light-activated ion channelswith light under suitable conditions to activate the polypeptide andopen the channel. As used herein the term “plurality” means more thanone.

According to certain aspects of the invention, the performance of a slowmutant molecule or plurality of the same or different slow mutantmolecules can be tuned for optimal use, including in the context oftheir use in conjunction with other light-activated ion channelmolecules or optical apparatus. For example, in order to achieve optimalcontrast for multiple-color stimulation, one may desire to eitherimprove or decrease the performance of one molecule with respect to oneanother, by the appendage of trafficking enhancing sequences or creationof genetic variants by site-directed mutagenesis, directed evolution,gene shuffling, or altering codon usage. Slow mutant molecules of theinvention and other light-activated ion channel molecules may haveinherently varying spectral sensitivity. This may be used to advantagein vivo (where scattering and absorption will vary with respect towavelength, coherence, and polarization), by tuning the linearity ornon-linearity of response to optical illumination with respect to time,power, and illumination history.

Certain embodiments of the invention include expression of 2, 3, 4, ormore different types of light-activated ion channel polypeptides, someor all of which are slow mutant polypeptides of the invention, inseparate subpopulations of a population of cells, which are referred toherein as “mixed” populations of cells. The subpopulations of cells canbe contacted with light in a manner that selectively activateslight-activated ion channels in one or more of the subpopulations ofcells but not necessarily activating each type of light-activated ionchannel in the mixed population. Certain embodiments of the inventioninclude expression of one type of slow mutant light-activated ionchannel polypeptide of the invention, in a population of cells, whichare referred to herein as “single” populations of cells. In someembodiments of the invention, a single or mixed population of cells isin culture and in certain embodiments of the invention a single or mixedpopulation of cells is in a subject.

Selection of suitable light and contact parameters to optimize thelight-activated ion channel open times of slow mutant and non-slowmutant light-activated ion channels in a mixed or single population canbe done using routine methods. Single and mixed populations of lightactivated ion channel polypeptides can be used in methods, assays,and/or treatment methods of the invention. In a mixed population,different light-activated ion channel polypeptides can be independentlyactivated by contacting the light-activated ion channel polypeptideswith different activating light parameters that are specific to eachtype of light-activated ion channel polypeptide.

A non-limiting example of a process to prepare and use a multi-lightactivated population of cells is as follows. A first light-activated ionchannel polypeptide that is a slow mutant polypeptide comprising anamino acid sequence set forth as SEQ ID NO: 1 is expressed in a firstsubpopulation of a population of cells. A second light-activated ionchannel that is not the slow mutant polypeptide is expressed in a secondsubpopulation of the population of cells, wherein the first and secondsubpopulations may be non-overlapping subpopulations or may beoverlapping subpopulations. The first light-activated ion channel andthe second light activated ion channel have ranges of activating lightwavelengths that do not entirely overlap. The population of cells iscontacted with a light under suitable conditions to activate the firstlight-activated ion channel polypeptides, for example: appropriate dosesof light, wavelength, pulse width, and power that activate the firstsubpopulation of cells, and the transmembrane voltage deflection ismeasured in a cell of the second subpopulation of cells contacted withthe first light test doses. The first light test dose parameters thatresults in activation of the first light activated ion channelpolypeptides and results in minimal (little or no) sub-thresholdtransmembrane voltage deflection in the second subpopulation of cells isdetermined. A similar process is used to identify suitable light doseparameters (such as: of light wavelength, pulse width, and power) thatactivate the second light-activated ion channels in the second subpopulation of cells but result in minimal (little or no) sub-thresholdtransmembrane voltage deflection in the first subpopulation of cells.Assays can be performed using such a population of cells, includingembodiments of methods in which the population of cells is contactedwith the first light test dose and the second light test dose determinedusing the steps above. The above-described process of optimizing lightdose parameters for multi-light activated ion channels, including butnot limited to slow mutant polypeptides, can be used to design andimplement assays that include slow mutant polypeptides of the invention,as well as other light-activated ion channels that are known in the art.

Treatment Methods

Some aspects of the invention include methods of treating a disorder orcondition in a cell, tissue, or subject using one or more slow mutantpolypeptides of the invention. Treatment methods of the invention mayinclude administering to a subject in need of such treatment, atherapeutically effective amount of a slow mutant molecule of theinvention to treat the disorder. It will be understood that in someaspects of the invention, a treatment administered to a subject is aprophylactic treatment, and in certain aspects of the invention atreatment is administered to a subject following diagnosis of a diseaseor condition in the subject. A treatment method of the invention mayreduce or eliminate a symptom or characteristic of a disorder, disease,or condition in a subject or may eliminate the disorder, disease, orcondition itself in the subject. It will be understood that a treatmentof the invention may reduce or eliminate progression of a disease,disorder or condition and may in some instances result in the regressionof the disease, disorder, or condition in a subject. A treatment neednot entirely eliminate the disease, disorder, or condition to beeffective. In some embodiments of the invention one or more slow mutantpolypeptides of the invention, non-limiting examples of which are SEQ IDNos: 1, 3, 11, 12, 14, 15, 17, 18, and functional variants thereof, maybe expressed in a cell population and used in methods to treat adisorder or condition.

Administration of a slow mutant polypeptide of the invention maybeperformed using various art-known methods. In some embodiments of theinvention, a vector that encodes a fusion protein comprising a slowmutant polypeptide of an invention is administered to a cell and/orsubject, resulting in the presence of the slow mutant polypeptide in acell in the subject. In certain aspects of the invention, a fusionprotein comprising a slow mutant polypeptide of the invention isadministered to a cell and/or subject, resulting in the presence of theslow mutant polypeptide in a cell in the subject. In some embodiments ofthe invention a cell comprising a slow mutant polypeptide or itsencoding nucleic acid is administered to a cell and/or subject,resulting in the presence of an expressed slow mutant polypeptide in acell in the subject. In some aspects of the invention, a slow mutantpolypeptide of the invention or its encoding nucleic acid isadministered as part of a pharmaceutical composition. A pharmaceuticalcomposition that comprises one or more of a vector comprising a nucleicacid that encodes a slow mutant polypeptide of the invention; a fusionprotein comprising a slow mutant polypeptide of the invention; or a cellthat comprises a slow mutant polypeptide of the invention or itsencoding nucleic acid of the invention, may be administered inembodiments of methods of the invention.

An effective amount of a slow mutant polypeptide or its encoding nucleicacid is an amount that increases the level of the slow mutantpolypeptide in a cell, tissue, or subject to a level that is beneficialfor the subject. An effective amount may also be determined by assessingphysiological effects of administration on a cell or subject, such as adecrease in symptoms of a disease or condition following administration.Art-known assays can also be employed to determine a level of a responseto a treatment of the invention. The amount of a treatment may be variedfor example by increasing or decreasing the amount of the slow mutantpolypeptide or encoding nucleic acid that is administered; by changingthe therapeutic composition in which the slow mutant polypeptide or itsencoding nucleic acid is administered, by changing the route ofadministration, by changing the dosage timing, by changing theactivation amounts and parameters of a slow mutant polypeptide of theinvention, and so on.

An effective amount of a slow mutant molecule for use in methods of theinvention, will vary with the particular condition being treated, theage and physical condition of the subject being treated; the severity ofthe condition, the duration of the treatment, the nature of theconcurrent therapy (if any), the specific route of administration, andthe like factors within the knowledge and expertise of the healthpractitioner. For example, an effective amount may depend upon thelocation and number of cells in the subject in which the slow mutantpolypeptide is to be expressed. An effective amount may also depend onthe location of the tissue to be treated. These factors are well knownand routinely determined for other therapeutic compounds, includingpreviously known light-activated ion channel molecules by those in theart and can be assessed the used to adjust treatment methods andadministration of a slow mutant molecule of the invention with no morethan routine experimentation. In some aspects of the invention a maximumdose of a composition to increase the level of a slow mutant polypeptideof the invention, and/or to alter the length or timing of activation ofa slow mutant polypeptide of the invention in a subject (alone or incombination with other therapeutic agents) be used, that is, the highestsafe dose or amount according to sound medical judgment. It will beunderstood by those in the art, however, that a patient/subject mayinsist upon a lower dose or tolerable dose for medical reasons,psychological reasons or for virtually any other reasons.

A slow mutant polypeptide of the invention, such as but not limited to:SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, 18, a functional variant of anythereof, or its encoding nucleic acid may be administered usingart-known methods and may be administered as part of a pharmaceuticalcomposition. Pharmaceutical compositions that can be used to administera slow mutant polypeptide of the invention or its encoding nucleic acidmay be administered alone, in combination with each other, and/or incombination with other drug therapies, or other treatment regimens thatare administered to subjects. A pharmaceutical composition used in someembodiments of methods of the invention comprise an effective amount ofa slow mutant molecule of the invention that will increase the level ofthe slow mutant polypeptide to a level that produces the desiredresponse in a unit of weight or volume suitable for administration to asubject.

The dose of a pharmaceutical composition that is administered to asubject to increase the level of a slow mutant polypeptide in a celland/or plurality of cells in the subject can be chosen in accordancewith different parameters, in particular in accordance with the mode ofadministration used and the state of the subject. Other factors includethe desired period of treatment. In the event that a response in asubject is insufficient at the initial doses applied, higher doses (oreffectively higher doses by a different and/or more localized deliveryroute) may be employed to the extent that patient tolerance permits. Theamount and timing of activation of a slow mutant polypeptide of theinvention (e.g., light wavelength, length of light contact, etc.) thathas been administered to a subject can also be adjusted based onefficacy of the treatment in a particular subject. Parameters forillumination and activation of a slow mutant polypeptide of theinvention that has been administered to a subject can be determinedusing information provided herein in conjunction with art-known methodsand without requiring undue experimentation.

Various modes of administration will be known to one of ordinary skillin the art that can be used to effectively deliver a pharmaceuticalcomposition to a subject to increase the level of a slow mutantpolypeptide of the invention in a desired cell, tissue or body region ofa subject. Methods for administering such a composition or otherpharmaceutical compound of the invention may be topical, intravenous,oral, intracavity, intrathecal, intrasynovial, buccal, sublingual,intranasal, transdermal, intravitreal, subcutaneous, intramuscular andintradermal administration. Delivery methods may include, but are notlimited to injection, microinjection, etc. The invention is not limitedby the particular modes of administration disclosed herein. Standardreferences in the art (e.g., Remington: The Science and Practice ofPharmacy, volumes I & II. Twenty-second edition, L. V. Allen, Jr,editor, Philadelphia, Pa.: Pharmaceutical Press. 2012) provide modes ofadministration and formulations for delivery of various pharmaceuticalpreparations and formulations in pharmaceutical carriers and meanstherein can be used in embodiments of methods of the invention. Otherart-known protocols may be used methods to administer a slow mutantmolecule of the invention, and in some embodiments of the invention, thedose amount, schedule of administration, sites of administration, modeof administration (e.g., intra-organ) and the like may vary from thosepresented herein.

Administration of a cell, vector, and/or slow mutant molecule of theinvention to increase a level of a slow mutant polypeptide of theinvention in an animal other than a human, and administration and use ofone or more slow mutant polypeptides of the invention for testingpurposes, veterinary therapeutic purposes, or other purposes innon-human animals may be carried out under substantially the sameconditions as described above. It will be understood by one of ordinaryskill in the art that this invention is applicable to both human andanimals. Thus, embodiments of the invention are contemplated for use inanimal husbandry and veterinary medicine as well as in humantherapeutics.

In some aspects of the invention, methods of treatment comprising use ofa slow mutant polypeptide of the invention are applied to cellsincluding but not limited to a neuronal cell, a nervous system cell, aneuron, a cardiac cell, a circulatory system cell, a visual system cell,an auditory system cell, a muscle cell, an immune system cell, anendocrine cell, etc.

Disorders, Diseases and Conditions

Slow mutant polypeptides of the invention may be expressed inpredetermined, preselected cell types, and activated to altervoltage-associated cell activities. In some aspects of the invention, aslow mutant polypeptide of the invention may be used to decrease the pHof a cell in which it is expressed. Such a technique may be used totreat alkalosis in a subject. Another aspect of the invention includesexpressing a slow mutant polypeptide of the invention in cell membraneand then activating the slow mutant polypeptide and generatingsub-cellular voltage or pH gradients, particularly at synapses and insynaptic vesicles to alter synaptic transmission, and mitochondria toimprove ATP synthesis.

In some embodiments, methods and slow mutants of the invention may beused for the treatment of visual system disorders, for example to treatvision reduction or loss and to increase visual function and ability ina subject. In a non-limiting example, a treatment method of theinvention may include administering a slow mutant polypeptide of theinvention to a subject known to have vision reduction or loss, and whenexpressed in a cell in in the subject the slow mutant polypeptidefunctions as a light-sensitive cell in the visual system, therebypermitting a gain of visual function in the subject. Other treatmentmethods are also included in the invention, such as, but not limited to,increasing auditory function in a subject, increasing memory function ina subject, reduction of one or more symptoms of a disease or disorder ina subject treated with one or more methods of the invention.

The present invention in some aspects, includes preparing nucleic acidsequences that encode slow mutant polypeptides of the invention,expressing the polypeptides encoded by the prepared nucleic acidsequences in cells and membranes; illuminating the cells and/ormembranes with light under suitable conditions to activate the slowmutant polypeptides, and producing rapid depolarization of the cellsand/or a change in conductance across the membrane in response to thelight, an extended open-time of the ion channel of the slow mutantpolypeptide after activation, versus a non-slow mutant parentlight-activated ion channel. In some aspects of the invention the cellsand/or membranes are in a subject. The ability to controllably altervoltage across membranes and cell depolarization with light has beendemonstrated as has the extended open-time that is a characteristic ofthe slow mutant polypeptides of the invention. The present inventionprovides novel ion channel polypeptides that provide longer open-timesand can be used in methods of light-control of cellular functions in invivo, in vitro, or ex vivo. Slow mutant polypeptides of the inventioncan be activated with lower amounts of light for activation of cells inwhich they are expressed than are useful in their non-mutant parentlight activated ion channel polypeptides and thus their use may be lessdamaging to cells and tissues in subjects and can also be used in arange of methods for drug screening, treatments, and researchapplications, some of which are describe herein.

In illustrative implementations of this invention, the ability tooptically perturb, modify, or control cellular function offers manyadvantages than is possible using previously known light-activated ionchannel polypeptides and their encoding nucleic acids. The presentinvention provides slow mutant polypeptides that when contacted withlight under suitable conditions: activate rapidly and maintain extendedopen-time. Slow mutant polypeptides of the invention are deliverableinto cells and subjects using routine methods and can be activated usinglevels of light that minimize cell and tissue damage from light. Thereagents use in the present invention (and the class of molecules thatthey represent), allow, at least: currents activated by lightwavelengths not useful in previous light-activated ion channels, lightactivated ion channels that when activated, permit effectively zerocalcium conductance, and different spectra from older molecules (openingup multi-color control of cells).

Non-limiting examples of disorders and conditions that can be treatedusing methods and slow mutant molecules of the invention include injury,brain damage, immune system conditions, cardiac conditions, cardiacdamage, muscle damage, muscle conditions, neurological conditions,degenerative neurological conditions, seizures, vision loss, hearingloss, etc. Diseases and conditions that may be treated using methods andslow mutants of the invention (for example those listed herein) comprisediseases and conditions characterized by abnormal electrical activity inone or more cells. Methods and slow mutant polypeptides of the inventioncan be expressed in such cells, for example in a subject having thedisease or condition, and illuminated with light under suitableconditions in a manner that alters and corrects abnormal electricalactivity in the cell or cells.

Non-limiting examples of neurological conditions include memory loss,memory disruption, learning disorders, depression, anxiety, seizuredisorders, etc. Non-limiting examples of degenerative neurologicalconditions include Parkinson's disease, Amyotrophic Lateral Sclerosis(ALS), Alzheimer's disease, etc.

EXAMPLES Example 1

Preparation and Testing of Single and Double Slow Mutant Polypeptides.

Embodiments of slow mutant molecules were prepared and tested. Resultsdemonstrated that the prepared embodiments of slow mutants resulted inslowing of photo-current decay after a brief activation with light.

Material and Methods

Whole-cell patch-clamp recordings were made using Multiclamp 700Bamplifier, a Digidata 1550A digitizer, and a PC running pClamp(Molecular Devices). For in vitro voltage clamp recordings, HEK cellswere patched lday post transfection and bathed in Tyrode solutioncontaining (in mM) 125 NaCl, 2 KCl, 3 CaCl₂, 1 MgCl₂, 10 HEPES, 30glucose, and with pH 7.3. Borosilicate glass pipettes (WarnerInstruments) with an outer diameter of 1.2 mm and a wall thickness of0.255 mm were pulled to a resistance of 3-7 MS2 with a P-97Flaming/Brown micropipette puller (Sutter Instruments) and filled with asolution containing (in mM) 135 K-gluconate, 8 NaCl, 0.1 CaCl₂, 0.6MgCl₂, 1 EGTA, 10 HEPES, 4 Mg-ATP, and 0.4 Na-GTP, and with pH 7.3 and290 mOsm. Cells were voltage clamped at −65 mV and illuminated by a 470nm LED (Lumencore) at 17.44 mW/mm² for 5 ms for photo-stimulation. Datawere analyzed using Clampfit (Molecular Devices) and Igor Pro(Wavemetrics).

Results

Two Chronos step function mutants were generated (FIGS. 2B and C).Chronos and two Chronos mutants (single mutant having Chronos sequencewith a C145S substitution and double mutant having Chronos sequence withC145S and D173A substitutions) were expressed in HEK cells, and thenresulting photo-currents produced by 470 nm light illumination wereexamined. Photo-currents were normalized to peak current amplitude forcomparison of channel kinetics. Normalized photo-currents were averagedfrom 3-4 cells each for Chronos and two mutants (FIG. 3A). As expectedChronos (control) had fastest decay of photo-current following 5 mphoto-illumination compare to the mutants (FIG. 3A). Single mutantphoto-current initially had faster decay than double mutant but itsdecay relaxed to similar to that of double mutant about 3 ms postillumination. Interestingly, a close-up examination of peak currents(FIG. 3B) revealed that photo-current activation was also significantlyslowed in the double mutant compare to Chronos and the single mutant.

Single (Chronos sequence with a C145S substitution) and double (Chronossequence with C145S and D173A substitutions) mutations were introducedinto a chronos polypeptide and the impact on activity of the expressedpolypeptide was determined. The single and double mutations altered theactivity of Chronos, which resulted in slowing photo-current decay witha brief photo-activation. Such manipulations can be used in testing andassessing neuron and excitable cell activity in disease, normal cells,in response to contact with compounds or under other conditions.

Example 2

Studies were performed that include preparing sequences and expressingslow mutant polypeptides in cells, tissues, and subjects. The expressedslow mutant polypeptides were selected from slow mutant polypeptides setforth herein as: SEQ ID No: 1, 3, 11, 12, 14, 15, 17, 18 and functionalvariants thereof. Some of the methods used in the studies are set forthExample 1 and others were routine methods in the art for preparingvectors, expressing fusion proteins, activating light-activated ionchannel polypeptides, measuring activity of light-activated ion channelpolypeptides, etc. General methods also applicable to light-activatedchannel molecules and methods used in studies of slow mutant moleculesof the invention are disclosed in publications such as US PublishedApplication No. 2010/0234273, US Published Application No. 20110165681,Chow B Y, et al., Methods Enzymol. 2011; 497:425-43; Chow, B Y, et al.Nature 2010 Jan. 7; 463(7277):98-102, the content of each of which isincorporated by reference herein.

Studies were performed to prepare sequences and to express slow mutantpolypeptides in cells, tissues, and subjects. Non-limiting exemplarymethods are set forth below. Vectors encoding slow mutant polypeptidemolecules were prepared using methods described in Example 1 and routinemethods were used to deliver the vectors into cells. Fusion proteinsthat were expressed comprised a slow mutant polypeptide such as one setforth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, or a functionalvariant of any of SEQ ID NO: 1, 3, 11, 12, 14, 15, 17, or 18, or a slowmutant polypeptide described elsewhere herein.

The slow mutant fusion proteins were expressed in one or more in vivo orin vitro cells. A cell in which the fusion protein was expressed wascontacted with suitable light to activate the expressed slow mutantpolypeptide and alter the electrical activity of the cell in which itwas expressed. The activation of the expressed slow mutant polypeptidealtered the electrical activity of the cell and in cells with abnormalelectrical activity, the activation reduced the abnormal electricalactivity of the cell.

Example 3

Experiments are performed in which sequences are prepared and used toexpress slow mutant polypeptides in cells, tissues, and subjects.Certain of the expressed slow mutant polypeptides are slow mutantpolypeptides set forth herein as SEQ ID No: 1, 3, 11, 12, 14, 15, 17, 18and functional variants thereof. Some of the methods used in the studiesare set forth Examples 1 and 2 and others are methods routinely used inthe art to prepare vectors, express fusion proteins, activatelight-activated ion channel polypeptides, measure activity oflight-activated ion channel polypeptides, etc. General methods alsoapplicable to light-activated channel molecules and methods that areused in studies of slow mutant molecules of the invention are disclosedin publications such as US Published Application No. 2010/0234273, USPublished Application No. 20110165681, Chow B Y, et al., MethodsEnzymol. 2011; 497:425-43; Chow, B Y, et al. Nature 2010 Jan. 7;463(7277):98-102, the content of each of which is incorporated byreference herein.

Slow mutant sequences are prepared and slow mutant polypeptides areexpressed in cells, tissues, and subjects. Some studies are set forthbelow. Slow mutant polypeptide molecules are prepared using methodsdescribed in Examples 1 and 2 and with routine methods. Experimentsinclude expressing fusion proteins comprising a slow mutant polypeptidesuch as one set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18,or a functional variant of any of SEQ ID NO: 1, 3, 11, 12, 14, 15, 17,or 18 as described elsewhere herein. Vectors are prepared that comprisea nucleic acid sequence that encodes one of SEQ ID NO: 1, 3, 11, 12, 14,15, 17, 18 or a functional variant thereof. Some of the vectors alsoinclude nucleic acids encoding one or more other polypeptides describedherein, such as but not limited to a trafficking polypeptide, an exportpolypeptide, a targeting polypeptide, etc.

Standard administration procedures are used to deliver vectors to cellsand subjects, the prepared vector is administered to a human or animalsubject who has a disease or condition, or is at risk of a disease orcondition, or is suspected of having a disease or condition thatincludes abnormal electrical activity in one or more cells or regions inthe subject.

The slow mutant fusion protein is expressed in a cell in the subject ina cell or region with abnormal electrical activity, the cell in whichthe fusion protein is expressed is contacted with suitable light toactivate the expressed slow mutant polypeptide and alter the electricalactivity of the cell in which it is expressed. The altered electricalactivity of the cell reduces the abnormal electrical activity of thecell. A disease in the subject that results from the abnormal electricalactivity is treated by contacting the expressed fusion protein withsuitable light.

Procedures are performed in which a described vector is administered toa subject having blindness and/or visual impairment that at least inpart, is the result of abnormal electrical activity in one or more of aneuronal cell and a visual system cell in the subject. Activation of theslow mutant polypeptide that is expressed treats and reduces theblindness in the subject. One or more of the symptoms and/orcharacteristics of the blindness and/or visual impairment being treatedwith the procedure is reduced in response to the procedure.

Procedures are performed in which a described vector, prepared usingmethods set forth in example 1, example 2, and/or using routineprocedures. The vector is administered to a subject having hearing lossor hearing impairment that results at least in part from abnormalelectrical activity in one or more of a neuronal cell and a auditorysystem cell in the subject. Activation of the slow mutant polypeptidethat is expressed treats one or more symptoms of hearing loss and/orhearing impairment and increases auditory function in the subject. Oneor more of the symptoms and/or characteristics of the hearing lossand/or hearing impairment being treated with the procedure are reducedin response to the procedure.

Additional procedures are performed in which a described vector isadministered to a subject having a seizure disorder or condition thatresults at least in part from abnormal electrical activity in one ormore of a neuronal cell in the brain of the subject. Activation of theslow mutant polypeptide that is expressed treats one or more symptoms ofseizure and reduces the seizures in the subject. One or more of thesymptoms and/or characteristics of the seizure disorder being treatedwith the procedure are reduced in response to the procedure.

Procedures are performed in which a described vector is administered toa subject having dementia, memory loss, Parkinson's disease, depression,ALS, and/or Alzheimer's disease symptoms that result at least in partfrom abnormal electrical activity in one or more of a neuronal cell inthe brain in the subject. The slow mutant polypeptide that is expressedis contacted with suitable light in an amount effective to decrease theabnormal electrical activity in the patient's cells that include thefusion protein and treats the dementia, memory loss, Alzheimer'sdisease, depression, Parkinson's disease, or ALS, and reduces one ormore symptoms and/or characteristics of the dementia, memory loss,Alzheimer's disease, depression, and Parkinson's disease, respectively,in the subject. One or more of the symptoms and/or characteristics ofthe dementia, memory loss, Alzheimer's disease, depression, ALS, and/orParkinson's disease being treated with the procedure are reduced inresponse to the procedure.

Procedures are performed in which a described vector is administered toa subject having a cardiac condition that results at least in part fromabnormal electrical activity in one or more of a cardiac cell, aneuronal cell, and a muscle cell in the subject. The slow mutantpolypeptide that is expressed is contacted with suitable light in anamount effective to reduce the abnormal electrical activity in thepatient's cells that include the fusion protein and to treat the cardiaccondition and to reduce one or more symptoms and/or characteristics ofthe cardiac condition in the subject. One or more of the symptoms and/orcharacteristics of the cardiac condition are reduced in response to theprocedure.

Procedures are performed in which a described vector is administered toa subject having an immune system condition that results at least inpart from abnormal electrical activity in one or more of an immunesystem cell in the subject. The slow mutant polypeptide that isexpressed is contacted with suitable light in an amount effective toreduce the abnormal electrical activity in the patient's cells thatinclude the fusion protein and to treat the immune system condition andto reduce one or more symptoms and/or characteristics of the immunesystem condition in the subject. One or more of the symptoms and/orcharacteristics of the immune system condition are reduced in responseto the procedure.

Procedures are performed in which a described vector is administered toa subject having a muscle defect, abnormal muscle activity, muscleassociated disease or condition that results at least in part fromabnormal electrical activity in a muscle cell in the subject or in acell that stimulates a muscle cell in the subject. The slow mutantpolypeptide that is expressed is contacted with suitable light in anamount effective to reduce the abnormal electrical activity in thepatient's cells that include the fusion protein and to treat the muscledefect, abnormal muscle activity, muscle associated disease or conditionand to reduce one or more symptoms and/or characteristics of the muscledefect, abnormal muscle activity, muscle associated disease or conditionthat results from abnormal electrical activity in cells in the subject.One or more of the symptoms and/or characteristics of the muscle defect,abnormal muscle activity, muscle associated disease or condition thatresults from abnormal electrical activity in cells in the subject arereduced in response to the procedure.

EQUIVALENTS

Although several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto; the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein.

In addition, any combination of two or more such features, systems,articles, materials, and/or methods, if such features, systems,articles, materials, and/or methods are not mutually inconsistent, isincluded within the scope of the present invention. All definitions, asdefined and used herein, should be understood to control over dictionarydefinitions, definitions in documents incorporated by reference, and/orordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.” The phrase“and/or,” as used herein in the specification and in the claims, shouldbe understood to mean “either or both” of the elements so conjoined,i.e., elements that are conjunctively present in some cases anddisjunctively present in other cases. Other elements may optionally bepresent other than the elements specifically identified by the “and/or”clause, whether related or unrelated to those elements specificallyidentified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications thatare cited or referred to in this application are incorporated byreference in their entirety herein. It is to be understood that themethods and compositions that have been described above are illustrativeapplications of the principles of the invention. Numerous modificationsmay be made by those skilled in the art without departing from the scopeof the invention. Although the invention has been described in detailfor the purpose of illustration, it is understood that such detail issolely for that purpose and variations can be made by those skilled inthe art without departing from the spirit and scope of the invention,which is defined by the following claims.

What is claimed:
 1. A light-activated ion channel polypeptide comprisingan amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, ora functional variant thereof.
 2. The light-activated ion channelpolypeptide of claim 1, comprising the amino acid sequence set forth asSEQ ID NO: 1 with 1, 2, 3, 4, or more amino acid sequence modifications,wherein a Serine (S) is present at the amino acid position thatcorresponds to amino acid 145 of SEQ ID NO: 1, and wherein thelight-activated ion-channel polypeptide has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to amino acids 61-295 of SEQ ID NO: 1 andat least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to theremaining amino acids in the sequence set forth as SEQ ID NO:
 1. 3. Thelight-activated ion channel polypeptide of claim 2, wherein the aminoacid sequence includes an Alanine (A) at the position corresponding toamino acid 173 of SEQ ID NO:
 1. 4. The light-activated ion channelpolypeptide of claim 1, wherein the light activated ion channelpolypeptide is comprises the amino acid sequence set forth as SEQ ID NO:3.
 5. (canceled)
 6. The light-activated ion channel polypeptide of claim1, wherein activating the light-activated ion channel polypeptide opensthe channel of the light-activated ion channel polypeptide, whereinactivating the ion channel polypeptide opens the ion channel of thelight-activated ion channel polypeptide, and wherein the channel remainsin an open state for a time period longer than an open state time periodof a control light-activated ion channel polypeptide.
 7. (canceled) 8.The light-activated ion channel polypeptide of claim 6, wherein thecontrol light-activated ion channel polypeptide is one of a Chronospolypeptide comprising the amino acid sequence set forth as SEQ ID NO:6, a Chrimson polypeptide comprising the amino acid sequence set forthas SEQ ID NO: 10, a CoChR polypeptide comprising the amino acid sequenceset forth as SEQ ID NO: 13, or a CsChR polypeptide comprising the aminoacid sequence set forth as SEQ ID NO:
 16. 9-10. (canceled)
 11. Thelight-activated ion channel polypeptide of claim 1, wherein thelight-activated ion channel polypeptide is expressed in a membrane, andoptionally the membrane is a cell membrane.
 12. (canceled)
 13. Thelight-activated ion channel polypeptide of claim 1, wherein thelight-activated ion channel polypeptide is expressed in a cell.
 14. Thelight-activated ion channel polypeptide of claim 12 or 13, wherein thecell is an excitable cell. 15-16. (canceled)
 17. The light-activated ionchannel polypeptide of claim 1, wherein activating the light-activatedion channel polypeptide alters the ion conductivity of the membrane inwhich the light-activated ion channel polypeptide is expressed. 18.(canceled)
 19. A fusion protein comprising the light-activated ionchannel polypeptide of claim
 1. 20. The fusion protein of claim 19,further comprising one or more of a trafficking polypeptide, a signalpolypeptide, an export polypeptide, and a detectable label polypeptide.21-42. (canceled)
 43. A nucleic acid sequence encoding a light-activatedion channel polypeptide of claim
 1. 44. (canceled)
 45. A vectorcomprising the nucleic acid sequence of claim
 43. 46-48. (canceled) 49.The vector of claim 45, wherein the vector is in a cell. 50-52.(canceled)
 53. A method of altering ion conductivity of a membrane, themethod comprising, expressing in a host membrane at least one of alight-activated ion channel polypeptide comprising an amino acidsequence set forth as SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or a functionalvariant thereof of and contacting the at least one of the expressedlight-activated ion channel polypeptides with a light that activates atleast one of the light-activated ion channels and alters the ionconductivity of the host membrane. 54-55. (canceled)
 56. The method ofclaim 53, wherein the host membrane is in a cell. 57-84. (canceled) 85.A method of assessing the effect of a candidate compound on ionconductivity of a membrane, the method comprising, (a) contacting a testmembrane comprising the light-activated ion channel polypeptidecomprising the amino acid sequence set forth as SEQ ID NO: 1, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ IDNO: 18, or a functional variant thereof with light under conditionssuitable for altering ion conductivity of the membrane; (b) contactingthe test membrane with a candidate compound; and (c) identifying thepresence or absence of a change in ion conductivity of the membranecontacted with the light and the candidate compound compared to ionconductivity in a control cell contacted with the light and notcontacted with the candidate compound; wherein a change in the ionconductivity in the test membrane compared to the control indicates aneffect of the candidate compound on the ion conductivity of the testmembrane.
 86. The method of claim 85, wherein the test membrane is in atest cell.
 87. The method of claim 86, wherein altering the ionconductivity of the test membrane depolarizes the test cell. 88-112.(canceled)